Journal of Cellular and Molecular Medicine

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1 Doxycycline reduces the migration of tuberous sclerosis complex (TSC)- null cells - effects on RhoA-GTPase and focal adhesion kinase (FAK) Journal: Journal of Cellular and Molecular Medicine Manuscript ID: JCMM R Wiley - Manuscript type: Original Article Date Submitted by the Author: n/a Complete List of Authors: Ng, Ho Yin; Woolcock Institute of Medical Research, Cell Biology; The University of Sydney, Pharmacology Oliver, Brian; Woolcock Institute of Medical Research, Cell Biology; The Univeristy of Sydney, Pharmacology Burgess, Janette; Woolcock Institute of Medical Research, Cell Biology; The Univeristy of Sydney, Pharmacology Krymskaya, Vera; University of Pennsylvania; University of Pennsylvania, Department of Medicine; University of Pennsylvania, Abramson Cancer Center Black, Judith; Woolcock Institute of Medical Research, ; The University of Sydney, Pharmacology Moir, Lyn; The University of Sydney, Pharmacology Keywords: pulmonary lymphangioleiomyomatosis, tuberous sclerosis gene complex-, doxycycline, rapamycin, wound closure, RhoA-GTPase, focal adhesion kinase, migration

2 Page of Journal of Cellular and Molecular Medicine Doxycycline reduces the migration of tuberous sclerosis complex (TSC)- null cells - effects on RhoA-GTPase and focal adhesion kinase (FAK) HY Ng,, BGG Oliver,, JK Burgess,, VP Krymskaya,, JL Black,, LM Moir,,*. Sydney Medical School, Discipline of Pharmacology, University of Sydney, Sydney, NSW, 00, Australia Cell Biology Group, Woolcock Institute of Medical Research, Sydney, NSW, 0, Australia Department of Medicine, University of Pennsylvania, Philadelphia, PA, United States of America Abramson Cancer Center, University of Pennsylvania, Philadelphia, PA, United States of America *Address for Correspondence: Dr. Lyn M. Moir, Woolcock Institute of Medical Research, PO Box M, Missenden Road, Camperdown, NSW, 00, Australia Ph: + 0, Fax: + 0, Running Title: Doxycycline inhibits migration of TSC-deficient cells B.G.G s current position is at the School of Medical and Molecular Biosciences, University of Technology Sydney, Sydney, NSW, Australia.

3 Page of Abstract Background and purpose: Lymphangioleiomyomatosis (LAM) is associated with dysfunction of the tuberous sclerosis complex (TSC) leading to enhanced cell proliferation and migration. This study aims to examine whether doxycycline, a tetracycline antibiotic, can inhibit the enhanced migration of TSC-deficient cells, identify signalling pathways through which doxycycline works and to assess the effectiveness of combining doxycycline with rapamycin (mammalian target of rapamycin complex [mtorc] inhibitor) in controlling cell migration, proliferation and wound closure. Experimental approach: TSC-positive and TSC-negative mouse embryonic fibroblasts (MEF), -TSC-positive and -TSC-null MEF and Eker rat uterine leiomyoma (ELT) cells were treated with doxycycline or rapamycin alone, or in combination. Migration, wound closure and proliferation were assessed using a transwell migration assay, time-lapse microscopy and manual cell counts respectively. RhoA-GTPase activity, phosphorylation of p0s kinase (p0sk) and focal adhesion kinase (FAK) in TSC-negative MEF treated with doxycycline were examined using enzyme-linked immunosorbent assay and immunoblotting techniques. Key results: The enhanced migration of TSC-null cells was reduced by doxycycline at concentrations as low as 0pM, while the rate of wound closure was reduced at µm. Doxycycline decreased RhoA-GTPase activity and phosphorylation of FAK in these cells but had no effect on the phosphorylation of p0sk, ERK/ or AKT. Combining doxycycline with rapamycin significantly reduced the rate of wound closure at lower concentrations than achieved with either drug alone. Conclusion and implications: This study shows that doxycycline inhibits TSC-null cell migration. Thus doxycycline has potential as an anti-migratory agent in the treatment of diseases with TSC dysfunction.

4 Page of Journal of Cellular and Molecular Medicine Key Words: pulmonary lymphangioleiomyomatosis, tuberous sclerosis gene complex-, doxycycline, rapamycin, migration, wound closure, RhoA-GTPase, focal adhesion kinase.

5 Page of Introduction Pulmonary lymphangioleiomyomatosis (LAM), a rare disease in women of childbearing age [,], is characterised by the irregular proliferation and migration of smooth muscle-like cells (LAM-cells) throughout the lungs, resulting in the obstruction of the small airways [,]. The disease ultimately manifests as cystic destruction of the lung parenchyma and the loss of pulmonary function []. LAM is associated with the mutational inactivation of the tuberous sclerosis gene complex (TSC), TSC TSC and TSC TSC [,]. Dysfunction of either TSC (hamartin) or TSC (tuberin) (with TSC being more common) results in enhanced cell proliferation and migration [-]. Over the last decade, knowledge of the underlying cellular mechanisms that drive LAM pathophysiology has been enhanced with an increased understanding of the rapamycin-sensitive, mammalian target of rapamycin complex (mtorc) [] and the rapamycin-insensitive mtorc signalling pathways [-]. The TSC/TSC complex indirectly regulates the phosphorylation of ribosomal p0s kinase (p0sk) and the initiation factor E-binding protein (E-BP) through the mtorc pathway, thus acting as a central regulator of cell growth and proliferation [-]. The functional TSC/TSC complex also acts to regulate the mtorc pathway, which controls actin cytoskeleton rearrangement through Rho GTPases, RhoA and Rac [,0]. Dysfunction of the TSC/TSC complex leads to the activation of mtorc, resulting in increased RhoA- GTPase activity and consequently enhanced cellular migration []. Cell migration in LAM has been demonstrated clinically, where it is reported that the same TSC mutation is observed in pulmonary LAM cells and angiomyolipoma (AML) cells []. This suggests that the cells have originated from a common origin, disseminating through the

6 Page of Journal of Cellular and Molecular Medicine vascular and lymphatic systems []. This also may explain the recurrence of LAM cells in the healthy lungs received by LAM patients post lung transplant [-]. In vitro studies by Goncharova et al. have also provided supporting evidence that a functional TSC/TSC complex acts to regulate normal cell migration through the mtorc pathway, which controls RhoA-GTPase activity []. The mtorc inhibitor rapamycin, a bacterial macrolide with immunosuppressive and antitumour properties, has been a major focus of LAM research. In vitro studies have shown rapamycin to effectively inhibit LAM cell proliferation [,,]. In addition, in the Multicenter International LAM Efficacy of Sirolimus (MILES) trial, McCormack et al. reported that patients with LAM treated with sirolimus (rapamycin) experienced stabilised lung function with a reduction in symptoms and improvement in quality of life compared to patients who were taking a placebo []. However limitations were present, most notably the continued decline in lung function following the discontinuation of sirolimus []. Similarly, in a murine model of TSC-null tumours, rapamycin inhibited tumour growth but its withdrawal resulted in TSC-null tumour regrowth together with a decreased survival []. Although the long term effects of rapamycin are not known in LAM, chronic sirolimus use has previously been associated with altered lipid and glucose metabolism resulting in hyperlipidemia, glucose intolerance, diabetes like syndromes and cancer [-]. Together, these in vitro and in vivo studies highlight that although rapamycin shows beneficial effects in the treatment of LAM, its requirement for long term use and its potential adverse effects highlight the need for alternative treatments. Since enhanced migration is a prominent feature of TSC-null cells, the lack of effect of rapamycin on migration [] suggests that mtorc inhibitors alone may not be the optimal

7 Page of treatment for LAM. Alternative treatments, in particular, drugs that tackle the increased migratory capacity of LAM cells may provide added benefit. Since LAM is a disease characterised by enhanced cell proliferation and migration, where both processes occur, the examination of the effects of drugs on both processes is necessary. Moses et al. demonstrated that doxycycline, a second-generation tetracycline antibiotic, improved lung function and quality of life in a single LAM patient, with minimal side-effects []. In addition, we have previously reported that doxycycline can reduce the mitochondrial activity and extracellular levels of active MMP- in human LAM cells and TSC-null mouse embryonic fibroblasts (MEF) []. Furthermore, doxycycline has been shown to inhibit migration of cells in the development of arterial intimal lesions [], breast carcinoma [] and human melanoma [0]. Since the loss of TSC plays a prominent role in the phenotypic characteristics in LAM, the study of cells that are well characterised for TSC dysfunction is highly relevant. Examples of these cells are the TSC knockout mouse embryonic fibroblasts (MEF) [], MEF in which TSC is stably reintroduced [] and the Eker rat uterine leiomyoma (ELT) cells in which TSC is naturally absent []. In this study, we investigated the effects of doxycycline on the migratory capacity of cells deficient for TSC, identified the signalling pathways through which doxycycline works and examined the potential of combining doxycycline with the mtorc inhibitor rapamycin, to inhibit the enhanced migration, proliferation and wound closure of TSC-deficient cells.

8 Page of Journal of Cellular and Molecular Medicine Materials and Methods Cell Culture Littermate derived p knockout TSC-negative MEF, TSC-positive MEF and isogenic p positive -TSC-null MEF and -TSC-positive MEF (where TSC was stably reintroduced) (a gift from Dr. D. Kwiatkowski, Brigham and Women s Hospital Boston, MA, USA) and Eker rat uterine leiomyoma (ELT) cells that were spontaneously deficient in TSC (a gift from Dr. Cheryl Walker, MD Anderson Cancer Center, TX, USA) were cultured as previously described [,,,,]. MEF, MEF and ELT cell preparation For experiments with MEF and MEF, the cells were seeded at a density of x cells.cm - in high glucose DMEM (Life Technologies, Carlsbad, CA, USA) containing % foetal bovine serum (FBS - Glendarach Biological, Melbourne, VIC, Australia) and % penicillin-streptomycin (Life Technologies) for hours. The medium was then changed to DMEM containing 0. % FBS and % penicillin-streptomycin (serum-reduced DMEM) for hours. The cells were then used as indicated in the experiments. ELT cells were cultured in DF medium (supplemented with % FBS) as previously described [,,,]. ELT cells were seeded at a density of x cells.cm - in DF medium for hours, after which the medium was changed to serum free DF-basal medium for hours as previously described []. The cells were then used as indicated in the experiments. Doxycycline, rapamycin and Y- preparation

9 Page of Doxycycline hyclate (Sigma-Aldrich, St. Louis, MO, USA) was freshly prepared in sterile distilled water at a stock concentration of 0 mm. Insolution rapamycin (Calbiochem, St. Louis, MO, USA) was diluted in sterile dimethyl sulfoxide (DMSO; Sigma-Aldrich) to a stock concentration of mm and stored at -0 C prior to use. Y- (Calbiochem) a ROCK inhibitor was diluted in sterile water to a stock concentration of mm and stored at - 0 C prior to use. For all experiments, doxycycline was used at a final concentration range of 00 fm - µm, rapamycin at nm (with respective DMSO vehicle controls) and Y- at 0. µm. For experiments in which the effects of combining doxycycline and rapamycin were assessed, various combinations of concentrations were used as indicated, with respective DMSO vehicle controls. Proliferation assay Cells that were seeded and placed under serum-reduced conditions in -well flat-bottom, tissue-culture treated polystyrene cell culture plates (BD Falcon, Becton Dickinson, Franklin Lakes, NJ, USA) as described above, were pretreated with doxycycline or rapamycin alone or in combination or with Y- in the presence of serum-reduced DMEM for minutes. The cells were subsequently stimulated with % FBS with or without drug for hours. Cells were then trypsinsed and proliferation was assessed by counting the number of viable cells (trypan blue exclusion) using a haemocytometer. Migration assay Cells that were seeded and placed in serum reduced conditions in cm tissue culture flasks (BD Falcon ) as described above were trypsinised, counted and resuspended in serum-

10 Page of Journal of Cellular and Molecular Medicine reduced DMEM. Cell migration was assessed using a transwell migration assay as previously described [,]. Briefly, cells in suspension were pre-treated with doxycycline or rapamycin alone or in combination, or with Y- (ROCK inhibitor) for minutes before seeding onto collagen type I ( µg/ml) coated cell culture inserts (.0 µm polyethylene terephthalate (PET) membrane (BD Falcon ) at a density of x cells per 0. cm. The cells were then allowed to migrate for hours towards a chemoattractant of % FBS at ⁰ C, % CO. Non-migrated cells were removed and migrated cells were fixed with % (v/v) paraformaldehyde (PFA), stained with 0. % (w/v) toludine blue (Sigma) containing 0. % (w/v) boric acid (Sigma) and imaged (Olympus BX0, Center Valley, PA, USA). Cell migration was measured by manually counting cells in five regions of 00 µm and expressed as an average. Wound Assay Cells that were seeded and placed under serum-reduced conditions in -well flat-bottom polystyrene cell culture plates precoated with collagen type I ( µg/ml) as described above, were pretreated with doxycycline or rapamycin alone, or in combination for minutes prior to wounding. A wound was created by scratching the cell layer with a sterile 00 µl pipette tip (Thermo Fisher Scientific, Waltham, MA, USA). The cells were rinsed with serumreduced DMEM to remove any cell debris and freshly prepared drug treatments were replaced into each well. The cells were then incubated at C, % CO in a humidified incubation chamber (Clear State Solutions, Victoria, Australia) mounted on a Nikon Eclipse Ti-microscope (Nikon Eclipse Ti, Tokyo, Japan). Time-lapse images were captured at intervals of hour for up to 0 hours as indicated and the rate of wound closure was assessed using Nikon Imaging software (NIS-Elements Imaging Software Version..0, Melville, NY, USA).

11 Page of Immunoblotting Levels of phosphorylated (phospho)-p0s kinase (p0sk), phospho-akt, phospho p/ MAPK (ERK/) and phospho-focal adhesion kinase (FAK) were measured using immunoblotting as previously described []. Briefly, cells that were seeded and placed in serum-reduced conditions in -well flat-bottom polystyrene cell culture plates were treated with doxycycline or rapamycin alone or in combination or with Y-. The cells were then lysed in modified RIPA buffer and the samples were size fractionated on % polyacrylamide gels and transferred to polyvinvylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA). The membranes were then blocked with % bovine serum albumin (BSA) diluted in phosphate buffered saline with 0.0% Tween-0 (PBS-T) and subsequently incubated with respective primary antibodies against phosphorylated proteins (as indicated in Table ) overnight at C, before incubation with respective immunoglobulin G- horseradish peroxidase (HRP) conjugated secondary antibody (as indicated in Table ) for hour at room temperature. The membranes were then visualised using chemiluminescence (Millipore, Temecula, CA, USA), imaged and analysed using a Kodak Image Station 000MM (Carestream Molecular Imaging Software Version.0.., Rochester, NY, USA). To allow for comparison of phosphorylated proteins to total proteins, antibodies were stripped from the membranes, the membranes were re-blocked and re-probed as previously described with either primary antibodies against total proteins (as indicated in Table ) or mouse anti-α-tubulin (loading control; :000 dilution in % PBS-T) (Santa Cruz Biotechnology, Santa Cruz, CA, USA) []. Respective immunoglobulin G- horseradish peroxidase (HRP) conjugated secondary antibodies (as indicated in Table ) were used.

12 Page of Journal of Cellular and Molecular Medicine RhoA Activation Assay RhoA activity was measured using the G-LISA RhoA Activation Assay Biochem Kit as per the manufacturer's instructions (Cytoskeleton, CO, USA). Briefly, cells that were seeded and placed in serum-reduced conditions in cm tissue cell culture flasks were treated with or without Y-, rapamycin or doxycycline alone or in combination as indicated for minute (as assessed by time-course experiments to exhibit maximum RhoA expression levels). The cells were then lysed and the activity of RhoA was measured using the G-LISA assay at an absorbance of 0 nm with the limits of detection at 0.0 ng. Following the manufacturer's instructions, levels of active RhoA were then normalised to total Rho (-A, -B, -C) levels as measured using immunoblotting (as detailed above). Mouse monoclonal anti- Rho (-A, -B, -C) primary antibody (diluted :000 - Millipore) and goat polyclonal antimouse immunoglobulin G- HRP conjugated secondary antibody (diluted :000 - Dako) were used. Statistical analysis Results from n experiments were analysed using Student's unpaired t-test, area under the curve, repeated measures one-way analysis of variance (ANOVA) or repeated measures twoway ANOVA with a Bonferroni post-test where appropriate (Graphpad Prism Version, Graphpad Software Inc, La Jolla, CA, USA). A probability (p) value of less than or equal to 0.0 was considered statistically significant.

13 Page of Results Enhanced cell migration and increased rate of wound closure in TSC-deficient cells In this study, we confirmed the enhanced migration of p knockout TSC-negative MEF compared to TSC-positive MEF (TSC-negative and TSC-positive, n=; p 0.0, Figure A) [,] and extended our findings to show, using -TSC-positive MEF in which TSC has been stably reintroduced, that cell migration was significantly lower in cells where TSC is present (-TSC-null and -TSC-positive MEF, n=; p 0.0, Figure A). In addition, the rate of wound closure in TSC-negative MEF was also significantly greater compared to TSC-positive MEF (area under the curve, TSC-negative and TSC-positive, n=; p 0.0, Figure B). Again, using -TSC-positive MEF, the rate at which the cells closed a wound was significantly decreased by the presence of TSC (-TSC-positive and -TSC-null, n=; p 0.0, Figure C). Doxycycline reduced enhanced cell migration in TSC-deficient cells Doxycycline (0 pm - µm) decreased % FBS-induced migration of TSC-negative MEF by % (TSC-negative, n=; p 0.0, Figure A) and of -TSC-null MEF by.. % (-TSC-null MEF, n=; p 0.0, Figure B). Doxycycline also decreased the rate of wound closure in both TSC-negative MEF and -TSC-null MEF ( µm - µm area under the curve, TSC-negative and -TSC-null MEF, n=; p 0.0, Figure C, D). Similarly, the ROCK inhibitor, Y- ( - µm) decreased FBS-induced migration of TSC-negative MEF by. -.0 % (TSC-negative MEF, n=; p 0.0, Figure E). In contrast, rapamycin, had no effect on the migration of TSC-negative MEF and -TSC-null MEF at any of the concentrations tested ( - 00 nm) (data not shown) in accordance with previous studies [].

14 Page of Journal of Cellular and Molecular Medicine Doxycycline and Y- had no effect on cell proliferation 0. % FBS and % FBS induced the proliferation of TSC-negative MEF to a greater extent than TSC-positive MEF (TSC-negative and TSC-positive, n=; p 0.0, Figure E) and this is in accordance with previous studies [,]. Cell proliferation of -TSC- positive MEF in the presence of % FBS was less than that in -TSC-null MEF (- TSC-positive and -TSC-null MEF, n=; p 0.0, Figure E). We have previously shown that doxycycline has no effect on the proliferation of TSC- positive or TSC-negative MEF [] and we were able to replicate these findings in - TSC-positive and -TSC-null MEF (-TSC-positive and -TSC-null MEF, n=; p>0.0, Figure E) and in ELT cells where doxycycline inhibited cell proliferation only at the highest concentration ( µm) (ELT cells, n=, p 0.0, Figure E). In addition, Y- had no effect on the proliferation of TSC-positive or TSC-negative MEF (TSC- positive and TSC-negative MEF, n=; p>0.0, Figure E). To confirm that proliferation in the above cell types can be inhibited, the mtorc inhibitor rapamycin was used as a positive control, showing a reduction in the proliferation of MEF, MEF and ELT cells (TSC-negative and TSC-positive, n=; p 0.0, Supplement Figure E, -TSC-positive and -TSC-null MEF, n=; p 0.0, Figure E and ELT, n=; p 0.0, Figure E). Furthermore, doxycycline and Y- had no effect on the phosphorylation of p0sk, (TSC-positive or TSC-negative, n=, p>0.0, doxycycline Figure A, B, Y- Figure C, D), whereas rapamycin inhibited the phosphorylation of p0sk (TSC-positive and TSC-negative, n=, p 0.0, Figure E, F). Doxycycline inhibits RhoA activity and reduces phosphorylation of FAK in TSC- negative MEF

15 Page of To investigate the mechanism through which doxycycline inhibits the migration of TSC- deficient cells, we examined the activity of RhoA-GTPase and the phosphorylation of FAK, AKT and ERK/. Under basal conditions and in the presence of % FBS, TSC-negative MEF exhibited increased RhoA-GTPase activity compared to TSC-positive MEF in accordance with published studies [,]. In addition, in TSC-positive MEF, RhoA- GTPase activity was induced in the presence of % FBS, whereas this was constitutively active in TSC-negative MEF (TSC-positive and TSC-negative MEF, 0. % FBS and % FBS, p 0.0, Figure A). Doxycycline ( µm & µm) and Y- ( µm) reduced elevated RhoA-GTPase activity in TSC-negative MEF by. -. % and. % at minute (TSC-negative, doxycycline and Y-, n=, p 0.0, Figure B), while rapamycin (00 nm) had no effect (TSC-negative, rapamycin 00 nm, n=, p 0.0, Figure B). In addition, doxycycline had no effect on RhoA-GTPase activity in TSC-positive MEF (TSC-positive, n=, p>0.0, Figure C). The amount of phospho-fak was constitutively higher in TSC-negative MEF compared to TSC-positive MEF (n=, p 0.0 Figure ). Doxycycline reduced elevated levels of phospho-fak in TSC-negative MEF while it had no effect in TSC-positive MEF (TSC- negative MEF, n=, p 0.0 Figure ). Doxycycline also had no effect on the levels of phospho-akt (n=, p>0.0, Figure A, B) or phospho-erk/ (p/) (n=, p>0.0, Figure C, D) in TSC-positive or TSC-negative MEF. Combined treatment of doxycycline and rapamycin inhibits the rate of wound closure in TSC-negative MEFs

16 Page of Journal of Cellular and Molecular Medicine We next assessed the effectiveness of combining rapamycin with doxycycline in inhibiting the proliferation, migration and the ability to close a wound of TSC-null MEF, as well as its effect on RhoA-GTPase activity. Doxycycline or rapamycin alone or in combination had no effect on the proliferation of TSC-positive MEF (TSC-positive, n=; p>0.0, Figure A). Combining doxycycline and rapamycin significantly inhibited TSC-negative MEF proliferation, migration and RhoA-GTPase activity, however the degree of inhibition was no greater compared to the individual drugs alone (Cell proliferation, TSC-negative, n=; p>0.0, Figure B, cell migration, TSC-negative, n=; p>0.0, Figure A and RhoA activity, n=, p>0.0, Figure B). The combination of doxycycline and rapamycin at sub-maximal concentrations decreased the rate of wound closure in TSC-negative MEF when compared to the individual effects of doxycycline or rapamycin alone (TSC-negative, n=; p<0.0, Figures C).

17 Page of Discussion A lack of treatment for LAM has prompted studies to further understand the pathogenic characteristics of the disease, in an attempt to pinpoint possible future therapeutic targets. In this study, we demonstrated the ability of doxycycline to reduce the enhanced migratory capacity of cells deficient for TSC through the inhibition of RhoA-GTPase activity and reduced levels of phospho-fak. In addition, we extended our research to examine the combination of doxycycline and rapamycin and showed this reduced the rate of wound closure and maintained the reduction in migration, proliferation and RhoA-GTPase activity in TSC-null cells. Since LAM is associated with TSC dysfunction, cells deficient for TSC such as MEF, - MEF and ELT cells, have been widely used to enhance our understanding of this disease []. The use of TSC-negative MEF and -TSC-null MEF in this study is of particular relevance to LAM as some LAM cells have been reported to be TSC negative [,,]. In addition, TSC-negative and -TSC-null MEF have also been shown to exhibit enhanced proliferation and migration, characteristics which are also shared by LAM cells [,,,0]. The dysfunction of TSC has been widely demonstrated to be associated with enhanced proliferation of LAM cells [,,,,,]. Many studies have focused on controlling this enhanced proliferation through the anti-proliferative properties of rapamycin (an mtorc inhibitor) [,,,]. However, it is as important to recognise that the loss of TSC function in LAM not only results in the hallmark manifestations of enhanced cell proliferation, but also enhanced cell migration. For this reason, we believe that targeting the increased migratory capacity of cells deficient for TSC may be beneficial.

18 Page of Journal of Cellular and Molecular Medicine It is well established that TSC plays a prominent role in the regulation of cell migration [,,,,]. Previous studies that examined cell migration using a transwell migration assay have demonstrated enhanced migratory capacity in cells deficient for TSC. In this study, we were able to confirm these findings and extended the study to assess the rate at which TSC-null cells can close a wound. Here, we demonstrated that the rate of wound closure in cells deficient for TSC was significantly increased compared to TSC-positive cells. In this study, we have demonstrated for the first time that doxycycline can reduce the migratory capabilities and reduce the rate of wound closure of TSC-negative MEF and - TSC-null MEF. It was of great interest that doxycycline inhibited cell migration at concentrations as low as 0 pm whereas it had no effect on the proliferation of -TSC- positive and -TSC-null MEF. This is in accordance with our previous study that showed doxycycline had no effect on the proliferation of TSC-positive, TSC-null MEF or human LAM cells []. Also in the present study, we showed that proliferation of ELT cells was inhibited by doxycycline only at high concentrations and these findings are similar to those of Chang et al. who reported that doxycycline at concentrations of - 0 µm [- µg.ml - ] had no effect on the proliferation of ELT cells but concentrations > µm [> µg.ml - ] decreased proliferation, increased apoptosis and altered cell morphology - effects which were due to doxycycline-induced toxicity []. Furthermore, we demonstrated rapamycin to be ineffective in the inhibition of migration and confirmed the findings of Goncharova et al. who showed rapamycin, at concentrations that significantly abrogated LAM cell proliferation, had no effect on human LAM cell migration []. Wound closure assays have previously been reported to reflect two crucial processes, cell proliferation and migration (Liang et al., 00). However, we have demonstrated in this study that, the ability of TSC-negative and -

19 Page of TSC-null MEFs to close a wound relies predominantly on migration and not proliferation, as rapamycin had little effect in these assays. Matrix metalloproteinase (MMP) - and - have often been associated with cell migration and invasion; however the concentrations of doxycycline that inhibited cell migration were significantly lower than those we have previously reported for effective MMP- inhibition []. This suggests that, although doxycycline is capable of MMP- inhibition, the reduction in migratory capacity demonstrated in this study is not likely to be due to the inhibition of MMPs. It was also interesting to observe that doxycycline was not as effective in the wound closure assay as in the transwell migration assay, where concentrations of only 0 µm [µg.ml - ] significantly inhibited wound closure. This may be in part due to other processes involved in the wound closure assay such as cell-cell interactions. The rapamycin-insensitive mtorc pathway regulates cell migration through RhoA- GTPase, and, in this study, we confirmed previous findings that RhoA-GTPase activity in TSC-negative MEF was higher than in TSC-positive MEF under basal conditions and in the presence of % FBS []. The mechanism of action through which doxycycline inhibits TSC-null cell migration was not known, however this study shows doxycycline inhibits the migration of TSC-null cells through the activity of RhoA-GTPase, whereas levels of phospho-p0sk, phospho-akt and phospho-erk/ were unaltered. However, the mechanism by which doxycycline inhibits cell migration at the lower concentrations at which RhoA-GTPase activity was not affected (0 pm - 00 nm), is likely to be due to targeting other signalling pathways.

20 Page of Journal of Cellular and Molecular Medicine Importantly, doxycycline inhibited the activity of RhoA-GTPase in TSC-negative MEF, while it was unaltered in TSC-positive MEF. RhoA-GTPase is known to play a prominent role in the regulation and organisation of the cytoskeleton by promoting the assembly of focal adhesions and by activating focal adhesion kinase (FAK) [,], processes which are important for cell motility. We have shown, in this study, that the protein tyrosine kinase FAK is constitutively active in TSC-negative MEF compared to TSC-positive MEF, thus supporting the finding of enhanced cell migration in TSC-negative MEF. Furthermore, doxycycline significantly inhibited levels of phospho-fak in TSC-negative MEF while they were unaltered in TSC-positive MEF. These data show that in TSC-negative MEF, doxycycline inhibited the activity of RhoA-GTPase at minute. This inhibition is then transduced downstream where FAK signalling was inhibited at hours and subsequently reduced functional migration at hours. The finding that doxycycline can regulate cell migration and FAK expression supports the findings by Sun et al. who showed doxycycline inhibited the adhesion and migration of melanoma cells through the inhibition of FAK expression []. Although no studies have demonstrated the sustained effects of doxycycline in the reduction of RhoA-GTPase activity, Sun et al. reported the effects of doxycycline in wound healing assays and FAK expression in the continued presence of doxycycline up to hours []. This suggests that the abrupt changes in RhoA-GTPase activity by doxycycline can influence outcomes of FAK expression and functional migration over a sustained period of time. It is now accepted that rapamycin alone may not be the most effective way to treat LAM, and investigations into combination therapies are underway [,]. Here, we explored the effects of combining doxycycline and rapamycin on the migration, proliferation, wound closure and RhoA activity of cells deficient for TSC. Although combining doxycycline and rapamycin

21 Page 0 of in this study inhibited those cellular parameters, the degree of inhibition was no greater than that of the individual drugs alone. The absence of a greater effect from the combination treatment was not surprising, as each drug works to inhibit independent functional characteristics and signalling pathways. However, it is important to note that the combination of doxycycline and rapamycin was not detrimental to the individual outcomes, i.e. it did not reduce the degree of inhibition caused by the individual drug treatment. Furthermore, as shown in the wound closure assay, combination treatment with submaximal doxycycline and rapamycin concentrations significantly reduced the rate of wound closure, whereas no effect was observed with individual doxycycline and rapamycin treatments at those concentrations. The mechanisms through which combination therapy reduced wound closure in TSC- negative MEF were not investigated in this study. The results from this study suggest that doxycycline may be of potential therapeutic benefit as a treatment for diseases in which TSC dysfunction results in enhanced cellular migration. In addition, this study provides preliminary evidence that the combination of doxycycline and rapamycin may lower the dosing requirement for rapamycin, and in turn potentially reduce the side effects associated with chronic rapamycin use. 0

22 Page of Journal of Cellular and Molecular Medicine Acknowledgements The authors would like to acknowledge the financial contributions from LAM Australasia Research Alliance (LARA), Cooperative Research Centre (CRC) for Asthma and Airways, The New Zealand LAM Charitable Trust, The LAM Foundation and NIH/NHLBIROHL00 (to VPK) B.G.G. Oliver was supported by a NHMRC Career Development Fellowship #0. J,K. Burgess was supported by a NHMRC Career Development Fellowship #. J.L. Black was supported by a NHMRC Senior Principal Fellowship #. We thank Dr. David Kwiatkowski for providing the MEFs and Dr. Cheryl Walker for providing the ELT cells. Conflicts of interest The authors have no conflict of interest to declare

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25 Page of Moir LM, Ng HY, Poniris MH, et al. Doxycycline inhibits matrix metalloproteinase- secretion from TSC-null mouse embryonic fibroblasts and lymphangioleiomyomatosis cells. Br J Pharmacol. 0; : -.. Bendeck MP, Conte M, Zhang M, et al. Doxycycline modulates smooth muscle cell growth, migration, and matrix remodeling after arterial injury. Am J Pathol. 00; 0: -.. Fife RS, Sledge GW, Jr. Effects of doxycycline on in vitro growth, migration, and gelatinase activity of breast carcinoma cells. J Lab Clin Med. ; : Seftor RE, Seftor EA, De Larco JE, et al. Chemically modified tetracyclines inhibit human melanoma cell invasion and metastasis. Clin Exp Metastasis. ; : -.. Zhang H, Bajraszewski N, Wu E, et al. PDGFRs are critical for PIK/Akt activation and negatively regulated by mtor. J Clin Invest. 00; : -.. Howe SR, Gottardis MM, Everitt JI, et al. Rodent model of reproductive tract leiomyomata. Establishment and characterization of tumor-derived cell lines. Am J Pathol. ; : -.. Goncharova E, Goncharov D, Noonan D, et al. TSC modulates actin cytoskeleton and focal adhesion through TSC-binding domain and the Rac GTPase. J Cell Biol. 00; : -.. Moir LM, Trian T, Ge Q, et al. Phosphatidylinositol -kinase isoform-specific effects in airway mesenchymal cell function. J Pharmacol Exp Ther. 0; : -.. Zhang H, Cicchetti G, Onda H, et al. Loss of Tsc/Tsc activates mtor and disrupts PIK-Akt signaling through downregulation of PDGFR. J Clin Invest. 00; : -.. Finlay GA, Malhowski AJ, Liu Y, et al. Selective inhibition of growth of tuberous sclerosis complex null cells by atorvastatin is associated with impaired Rheb and Rho GTPase function and reduced mtor/s kinase activity. Cancer Res. 00; : -.. Kwiatkowski DJ. Animal models of lymphangioleiomyomatosis (LAM) and tuberous sclerosis complex (TSC). Lymphat Res Biol. 0; : -.. Hirama M, Atsuta R, Mitani K, et al. Lymphangioleiomyomatosis diagnosed by immunocytochemical and genetic analysis of lymphangioleiomyomatosis cell clusters found in chylous pleural effusion. Intern Med. 00; : -.. Darling TN, Pacheco-Rodriguez G, Gorio A, et al. Lymphangioleiomyomatosis and TSC-/- cells. Lymphat Res Biol. 0; : Goncharova EA, Ammit AJ, Irani C, et al. PIK is required for proliferation and migration of human pulmonary vascular smooth muscle cells. Am J Physiol Lung Cell Mol Physiol. 00; : L-.. Yu JJ, Robb VA, Morrison TA, et al. Estrogen promotes the survival and pulmonary metastasis of tuberin-null cells. Proc Natl Acad Sci U S A. 00; : -0.. Yu J, Parkhitko AA, Henske EP. Mammalian target of rapamycin signaling and autophagy: roles in lymphangioleiomyomatosis therapy. Proc Am Thorac Soc. 0; : -.. Kwiatkowski DJ, Manning BD. Tuberous sclerosis: a GAP at the crossroads of multiple signaling pathways. Hum Mol Genet. 00; Spec No. : R-.

26 Page of Journal of Cellular and Molecular Medicine Lesma E, Eloisa C, Isaia E, et al. Development of a lymphangioleiomyomatosis model by endonasal administration of human TSC-/- smooth muscle cells in mice. Am J Pathol. 0; : -0.. Chang WY, Clements D, Johnson SR. Effect of doxycycline on proliferation, MMP production, and adhesion in LAM-related cells. Am J Physiol Lung Cell Mol Physiol. 0; : L-00.. Chen CS, Alonso JL, Ostuni E, et al. Cell shape provides global control of focal adhesion assembly. Biochem Biophys Res Commun. 00; : -.. Lamb RF, Roy C, Diefenbach TJ, et al. The TSC tumour suppressor hamartin regulates cell adhesion through ERM proteins and the GTPase Rho. Nat Cell Biol. 000; : -.. Sun T, Zhao N, Ni CS, et al. Doxycycline inhibits the adhesion and migration of melanoma cells by inhibiting the expression and phosphorylation of focal adhesion kinase (FAK). Cancer Lett. 00; : -0.

27 Page of Figure Legends Figure : % FBS-induced migration of A. TSC-positive (n=) and TSC-negative (n=) MEF and -TSC-positive MEF (n=) and -TSC-null MEF (n=). Datarepresent the average number of cells migrated in five regions of 00 µm ± SEM *p 0.0 unpaired t-test. Wound closure assay of B. TSC-positive (black line, n=) and TSC-negative MEF (red line, n=) over 0 hours and C. -TSC-positive (black line, n=) and -TSC-null MEF (red line, n=) over 0 hours. Data expressed as a percentage closure ± SEM in response to0. % FBS stimulation *p 0.0 repeated measures one-way ANOVA. Figure : Migration of A. TSC-negative MEF (n=) and B. -TSC-null MEF following minutes pretreatment with doxycycline. Data expressed as the average number of cells migrated in five regions of 00 µm ± SEM *p 0.0 repeated measures one-way ANOVA. Wound closure assay of C. TSC-negative MEF (n=) and D. -TSC-null MEF (n=) following minutes pretreatment with doxycycline. Data expressed as area under the curve (AUC) over 0 hours ± SEM. *p 0.0 repeated measures one-way ANOVA. Migration of E. TSC-negative MEF (n=) following minutes pretreatment with Y-. Data expressed as the average number of cells migrated per 00 µm *p 0.0 repeated measures one-way ANOVA. Figure : Proliferation of A. TSC-positive MEF (n=) and B. TSC-negative MEF (n=) treated with doxycycline, vehicle or rapamycin alone, or in combination as indicated. Data expressed as mean ± SEM *p 0.0 repeated measures one-way ANOVA compared to FBS or DMSO vehicle control where appropriate. Bonferroni post-test was used.

28 Page of Journal of Cellular and Molecular Medicine Figure : Phospho-p0SK levels in A. TSC-positive MEF (n=) and B. TSC-negative MEF (n=) in the presence (+) or absence (-) of doxycycline as indicated, C. TSC-positive MEF (n=) and D. TSC-negative MEF (n=) in the presence (+) or absence (-) of Y- as indicated and E. TSC-positive MEF (n=) and F. TSC-negative MEF (n=) in the presence (+) or absence (-) of vehicle control (V) or rapamycin as indicated. Representative western blots of phospho-p0sk, p0sk and α-tubulin are shown (A-F) above mean data. Data expressed as mean ± SEM *p 0.0 repeated measures one-way ANOVA with a Bonferroni post-test. Figure : RhoA-GTPase activity in A. TSC-positive (n=) and TSC-negative MEF (n=) under basal conditions (0. % FBS) and in the presence of the stimulus % FBS, B. TSC- negative MEF (n=) following minute of treatment with doxycycline, DMSO vehicle control, rapamycin or Y- as indicated and C. TSC-positive MEF (n=) following minute of treatment with doxycycline, or Y-. RhoA activity corrected for total Rho (-A, -B, -C) and expressed as a percentage of % FBS ± SEM *p 0.0 repeated measures oneway ANOVA with a Bonferroni post-test. Figure : Phospho-FAK levels in TSC-positive and TSC-negative MEF (n=) in the presence (+) or absence (-) of doxycycline treatment for hours. Representative western blots of phospho-fak, FAK and α-tubulin are shown above mean data. Data expressed as mean ± SEM *p 0.0 repeated measures one-way ANOVA with a Bonferroni post-test. Figure : Phospho-AKT levels in A. TSC-positive MEF (n=) and B. TSC-negative MEF (n=) and phospho-erk/ (p/) levels in C. TSC-positive MEF (n=) and D. TSC- negative MEF (n=) in the presence (+) or absence (-) of doxycycline as indicated.

29 Page of Representative western blots of phospho-akt, AKT, phospho-erk/ (p/), ERK/ (p/) and α-tubulin are shown above mean data. Data expressed as mean ± SEM *p 0.0 repeated measures one-way ANOVA with a Bonferroni post-test. Figure : Proliferation of A. TSC-positive MEF (n=), B. TSC-negative MEF (n=) and C. cell migration of TSC-negative MEF (n=) treated with doxycycline (D) at nm, vehicle (V) or rapamycin (R) at nm alone, or in combination (RD). Data expressed as mean ± SEM *p 0.0 repeated measures one-way ANOVA. D. RhoA activity of TSC-negative MEF (n=) treated with doxycycline nm, vehicle or rapamycin nm alone or in combination. RhoA activity corrected for total Rho (-A, -B, -C) and expressed as a percentage of % FBS ± SEM *p 0.0 repeated measures one-way ANOVA. E. Wound closure of TSC- negative MEF (n=) treated with doxycycline at nm, vehicle or rapamycin nm alone or in combination. Data expressed as area under the curve (AUC) over 0 hours ± SEM. *p 0.0 repeated measures one-way ANOVA compared to FBS or DMSO vehicle control (V) where appropriate. Supplement Figure E: Proliferation of TSC-positive (n=) and TSC-negative MEF (n=) treated with vehicle (V) or rapamycin. Data expressed as mean ± SEM # p 0.0 TSC- positive MEF compared to % FBS and *p 0.0 TSC-negative MEF compared to % FBS, repeated measures two-way ANOVA compared to DMSO vehicle control (V). Supplement Figure E. Proliferation of -TSC-positive MEF (n=) and -TSC-null MEF (n=) treated with vehicle (V) or rapamycin. Data expressed as mean ± SEM # p 0.0 -TSC-positive MEF compared to % FBS and *p 0.0 -TSC-null MEF

30 Page of Journal of Cellular and Molecular Medicine compared to % FBS, repeated measures two-way ANOVA compared to DMSO vehicle control (V). Supplement Figure E. Proliferation of ELT cells (n=) treated with vehicle (V) or rapamycin. Data expressed as mean ± SEM *p 0.0 repeated measures two-way ANOVA compared to DMSO vehicle control (V). Supplement Figure E. Basal level (0. % FBS) and % FBS-induced proliferation of TSC-positive (n=) and TSC-negative (n=) MEF. Data expressed as mean ± SEM *p 0.0 repeated measures two-way ANOVA. Supplement Figure E. Basal level (0. % FBS) and % FBS-induced proliferation of -TSC-positive (n=) and -TSC-null MEF (n=). Data expressed as mean ± SEM *p 0.0 repeated measures two-way ANOVA. Supplement Figure E. Proliferation of -TSC-positive (n=) and -TSC-null MEF (n=), D. ELT cells treated with doxycycline. Data expressed as mean ± SEM *p 0.0 repeated measures one-way ANOVA. Supplement Figure E. Proliferation of TSC-positive (n=) and TSC-negative MEF (n=) treated with Y-. Data expressed as mean ± SEM *p 0.0 repeated measures oneway ANOVA. Supplement Figure E. Cell migration of TSC-negative MEF treated with doxycycline, vehicle or rapamycin alone, or in combination as indicated. Data expressed as mean ± SEM

31 Page of *p 0.0 repeated measures one-way ANOVA compared to FBS or DMSO vehicle control where appropriate. Bonferroni post-test was used. Supplement Figure E. RhoA activity in TSC-negative MEF (n=) treated with doxycycline, vehicle or rapamycin alone, or in combination as indicated. RhoA activity corrected for total Rho (-A, -B, -C) and expressed as a percentage of % FBS *p 0.0 repeated measures one-way ANOVA compared to FBS or DMSO vehicle control where appropriate. Bonferroni post-test was used. Supplement Figure E. Wound closure of TSC-negative MEF (n=) treated with doxycycline, vehicle or rapamycin alone, or in combination as indicated. Data expressed as percentage closure over 0 hours *p 0.0 repeated measures one-way ANOVA compared to FBS or DMSO vehicle control where appropriate. Bonferroni post-test was used. Figure Legends Figure : % FBS-induced migration of A. TSC-positive (n=) and TSC-negative (n=) MEF and -TSC-positive MEF (n=) and -TSC-null MEF (n=). Data represent the average number of cells migrated in five regions of 00 µm ± SEM *p 0.0 unpaired t-test. Wound closure assay of B. TSC-positive (black line, n=) and TSC- negative MEF (red line, n=) over 0 hours and C. -TSC-positive (black line, n=) and -TSC-null MEF (red line, n=) over 0 hours. Data expressed as a percentage closure ± SEM in response to0. % FBS stimulation *p 0.0 repeated measures one-way ANOVA with a Bonferroni post test.

32 Page of Journal of Cellular and Molecular Medicine Figure : Migration of A. TSC-negative MEF (n=) and B. -TSC-null MEF following minutes pretreatment with doxycycline. Data expressed as the average number of cells migrated in five regions of 00 µm ± SEM. Wound closure assay of C. TSC-negative MEF (n=) and D. -TSC-null MEF (n=) following minutes pretreatment with doxycycline. Data expressed as area under the curve (AUC) over 0 hours ± SEM.. Migration of E. TSC- negative MEF (n=) following minutes pretreatment with Y-. Data expressed as the average number of cells migrated per 00 µm *p 0.0 repeated measures one-way ANOVA compared with FBS with a Bonferroni post-test. Figure : Phospho-p0SK levels in A. TSC-positive MEF (n=) and B. TSC-negative MEF (n=) in the presence (+) or absence (-) of doxycycline as indicated, C. TSC-positive MEF (n=) and D. TSC-negative MEF (n=) in the presence (+) or absence (-) of Y- as indicated and E. TSC-positive MEF (n=) and F. TSC-negative MEF (n=) in the presence (+) or absence (-) of vehicle control (V) or rapamycin as indicated. Representative western blots of phospho-p0sk, p0sk and α-tubulin are shown (A-F) above mean data. Data expressed as mean ± SEM *p 0.0 repeated measures one-way ANOVA with a Bonferroni post-test (compared with % FBS). Figure : RhoA-GTPase activity in A. TSC-positive (n=) and TSC-negative MEF (n=) under basal conditions (0. % FBS) and in the presence of the stimulus % FBS # p<0.0 TSC-positive vs TSC-negative cells, B. TSC-negative MEF (n=) following minute of treatment with doxycycline, vehicle control (DMSO), rapamycin or Y- as indicated and C. TSC-positive MEF (n=) following minute of treatment with doxycycline, or Y-. RhoA activity corrected for total Rho (-A, -B, -C) and expressed as a percentage of %

33 Page of FBS ± SEM *p 0.0 repeated measures one-way ANOVA with a Bonferroni post-test (compared with % FBS). Figure : Phospho-FAK levels in TSC-positive and TSC-negative MEF (n=) in the presence (+) or absence (-) of doxycycline treatment for hours. Representative western blots of phospho-fak, FAK and α-tubulin are shown above mean data. Data expressed as mean ± SEM #p<0.0 compared with TSC-negative cells; *p 0.0 repeated measures oneway ANOVA with a Bonferroni post-test (compared with % FBS). Figure : Phospho-AKT levels in A. TSC-positive MEF (n=) and B. TSC-negative MEF (n=) and phospho-erk/ (p/) levels in C. TSC-positive MEF (n=) and D. TSC- negative MEF (n=) in the presence (+) or absence (-) of doxycycline as indicated. Representative western blots of phospho-akt, AKT, phospho-erk/ (p/), ERK/ (p/) and α-tubulin are shown above mean data. Data expressed as mean ± SEM *p 0.0 repeated measures one-way ANOVA with a Bonferroni post-test (comparison with % FBS). Figure : Proliferation of A. TSC-positive MEF (n=) and B. TSC-negative MEF (n=) treated with doxycycline, vehicle or rapamycin alone, or in combination as indicated. Data expressed as mean ± SEM *p 0.0 repeated measures one-way ANOVA compared to FBS #p<0.0 repeated measures one-way ANOVA compared to Vehicle (DMSO) control where appropriate. Bonferroni post-test was used. Figure : Cell migration of A. TSC-negative MEF (n=), treated with doxycycline (D) vehicle or rapamycin (R) alone, or in combination (R+D). Data expressed as mean ± SEM

34 Page of Journal of Cellular and Molecular Medicine *p 0.0 comparison with % FBS, $ p<0.0 comparison with vehicle control, using repeated measures one-way ANOVA. B. RhoA activity of TSC-negative MEF (n=) treated with doxycycline(d), vehicle or rapamycin (R) alone or in combination. RhoA activity corrected for total Rho (-A, -B, -C) and expressed as a percentage of % FBS ± SEM *p 0.0 comparison with % FBS, $ p<0.0 comparison with vehicle control using repeated measures one-way ANOVA. C. Wound closure of TSC-negative MEF (n=) treated with doxycycline, vehicle or rapamycin nm alone or in combination. Data expressed as area under the curve (AUC) over 0 hours ± SEM. *p 0.0 comparison with % FBS, $ p<0.0 comparison with vehicle control, analysed using repeated measures one-way ANOVA as appropriate. Supplement Figure E. Basal level (0. % FBS) and % FBS-induced proliferation of TSC-positive (n=) and TSC-negative (n=) MEF. Data expressed as mean ± SEM *p 0.0 repeated measures two-way ANOVA with a Bonferroni post test. Supplement Figure E. Basal level (0. % FBS) and % FBS-induced proliferation of -TSC-positive (n=) and -TSC-null MEF (n=). Data expressed as mean ± SEM *p 0.0 repeated measures two-way ANOVA with a Bonferroni post test. Supplement Figure E. Proliferation of -TSC-positive (n=) and -TSC-null MEF (n=), treated with doxycycline. Data expressed as mean ± SEM *p 0.0 repeated measures one-way ANOVA with a Bonferroni post test.

35 Page of Supplement Figure E. Proliferation of ELT cells treated with doxycycline. Data expressed as mean ± SEM *p 0.0 compared to % FBS using repeated measures one-way ANOVA with a Bonferroni post test. Supplement Figure E. Proliferation of TSC-positive (white bars, n=) and TSC-negative MEF (grey bars, n=), treated with Y-. Data expressed as mean ± SEM *p 0.0 repeated measures one-way ANOVA with a Bonferroni post test. Supplement Figure E: Proliferation of TSC-positive (n=) and TSC-negative MEF (n=) treated with vehicle (V) or rapamycin. Data expressed as mean ± SEM # p 0.0 TSC- positive MEF compared to % FBS and *p 0.0 TSC-negative MEF compared to % FBS, repeated measures two-way ANOVA compared to DMSO vehicle control (V) with a Bonferroni post test. Supplement Figure E. Proliferation of -TSC-positive MEF (n=) and -TSC-null MEF (n=) treated with vehicle (V) or rapamycin. Data expressed as mean ± SEM # p 0.0 -TSC-positive MEF compared to % FBS and *p 0.0 -TSC-null MEF compared to % FBS, repeated measures two-way ANOVA compared to DMSO vehicle control (V) with a Bonferroni post test. Supplement Figure E. Proliferation of ELT cells treated with vehicle (V) or rapamycin. Data expressed as mean ± SEM # p 0.0 ELT cells compared to % FBS repeated measures one-way ANOVA compared to DMSO vehicle control (V) with a Bonferroni post test.

36 Page of Journal of Cellular and Molecular Medicine Table. Antibodies for immunoblotting Target Protein p0sk AKT p/ ERK/ MAPK FAK α-tubulin Primary Antibody / Concentration (Supplier) Rabbit polyclonal anti-mouse Phospho-p0SK (Thr ) / :000 (Cell Signalling Technology, Danvers, MA, USA) Rabbit polyclonal anti-mouse p0sk / :000 (Cell Signalling Technology) Rabbit monoclonal anti-mouse Phospho-AKT (Thr ) / :000 Secondary Antibody (Supplier) Goat polyclonal anti-rabbit immunoglobulin G- HRP conjugated :000 (Dako Glosturp, Denmark) Treatment Incubation Time (hrs) (Cell Signalling Technology,) / Rabbit monoclonal anti-mouse AKT / :000 (Cell Signalling Technology,) Rabbit monoclonal anti-mouse Phospho-p/ MAPK (ERK/) (Thr 0 /Thr 0 ) / :000 (Cell Signalling Technology,) Rabbit monoclonal anti-mouse p/ MAPK (ERK/) / :000 (Cell Signalling Technology,) Rabbit polyclonal anti-mouse Phospho-FAK (Tyr ) / :000 (Cell Signalling Technology,) Rabbit polyclonal anti-mouse FAK / :000 (Cell Signalling Technology,) Mouse polyclonal α-tubulin :000 (SantaCruz) Goat polyclonal anti-mouse immunoglobulin G- HRP conjugated :000 (Dako) *Primary and secondary antibodies were diluted in % BSA/PBS-T. / / -

37 Page of Doxycycline reduces the migration of tuberous sclerosis complex (TSC)- null cells - effects on RhoA-GTPase and focal adhesion kinase (FAK) HY Ng,, BGG Oliver,, JK Burgess,, VP Krymskaya,, JL Black,, LM Moir,,*. Sydney Medical School, Discipline of Pharmacology, University of Sydney, Sydney, NSW, 00, Australia Cell Biology Group, Woolcock Institute of Medical Research, Sydney, NSW, 0, Australia Department of Medicine, University of Pennsylvania, Philadelphia, PA, United States of America Abramson Cancer Center, University of Pennsylvania, Philadelphia, PA, United States of America *Address for Correspondence: Dr. Lyn M. Moir, Woolcock Institute of Medical Research, PO Box M, Missenden Road, Camperdown, NSW, 00, Australia Ph: + 0, Fax: + 0, Running Title: Doxycycline inhibits migration of TSC-deficient cells B.G.G s current position is at the School of Medical and Molecular Biosciences, University of Technology Sydney, Sydney, NSW, Australia.

38 Page of Journal of Cellular and Molecular Medicine Abstract Background and purpose: Lymphangioleiomyomatosis (LAM) is associated with dysfunction of the tuberous sclerosis complex (TSC) leading to enhanced cell proliferation and migration. This study aims to examine whether doxycycline, a tetracycline antibiotic, can inhibit the enhanced migration of TSC-deficient cells, identify signalling pathways through which doxycycline works and to assess the effectiveness of combining doxycycline with rapamycin (mammalian target of rapamycin complex [mtorc] inhibitor) in controlling cell migration, proliferation and wound closure. Experimental approach: TSC-positive and TSC-negative mouse embryonic fibroblasts (MEF), -TSC-positive and -TSC-null MEF and Eker rat uterine leiomyoma (ELT) cells were treated with doxycycline or rapamycin alone, or in combination. Migration, wound closure and proliferation were assessed using a transwell migration assay, time-lapse microscopy and manual cell counts respectively. RhoA-GTPase activity, phosphorylation of p0s kinase (p0sk) and focal adhesion kinase (FAK) in TSC-negative MEF treated with doxycycline were examined using enzyme-linked immunosorbent assay and immunoblotting techniques. Key results: The enhanced migration of TSC-null cells was reduced by doxycycline at concentrations as low as 0pM, while the rate of wound closure was reduced at µm. Doxycycline decreased RhoA-GTPase activity and phosphorylation of FAK in these cells but had no effect on the phosphorylation of p0sk, ERK/ or AKT. Combining doxycycline with rapamycin significantly reduced the rate of wound closure at lower concentrations than achieved with either drug alone. Conclusion and implications: This study shows that doxycycline inhibits TSC-null cell migration. Thus doxycycline has potential as an anti-migratory agent in the treatment of diseases with TSC dysfunction.

39 Page of Key Words: pulmonary lymphangioleiomyomatosis, tuberous sclerosis gene complex-, doxycycline, rapamycin, migration, wound closure, RhoA-GTPase, focal adhesion kinase.

40 Page of Journal of Cellular and Molecular Medicine Introduction Pulmonary lymphangioleiomyomatosis (LAM), a rare disease in women of childbearing age [,], is characterised by the irregular proliferation and migration of smooth muscle-like cells (LAM-cells) throughout the lungs, resulting in the obstruction of the small airways [,]. The disease ultimately manifests as cystic destruction of the lung parenchyma and the loss of pulmonary function []. LAM is associated with the mutational inactivation of the tuberous sclerosis gene complex (TSC), TSC and TSC [,]. Dysfunction of either TSC (hamartin) or TSC (tuberin) (with TSC being more common) results in enhanced cell proliferation and migration [-]. Over the last decade, knowledge of the underlying cellular mechanisms that drive LAM pathophysiology has been enhanced with an increased understanding of the rapamycinsensitive, mammalian target of rapamycin complex (mtorc) [] and the rapamycininsensitive mtorc signalling pathways [-]. The TSC/TSC complex indirectly regulates the phosphorylation of ribosomal p0s kinase (p0sk) and the initiation factor E-binding protein (E-BP) through the mtorc pathway, thus acting as a central regulator of cell growth and proliferation [-]. The functional TSC/TSC complex also acts to regulate the mtorc pathway, which controls actin cytoskeleton rearrangement through Rho GTPases, RhoA and Rac [,0]. Dysfunction of the TSC/TSC complex leads to the activation of mtorc, resulting in increased RhoA-GTPase activity and consequently enhanced cellular migration []. Cell migration in LAM has been demonstrated clinically, where it is reported that the same TSC mutation is observed in pulmonary LAM cells and angiomyolipoma (AML) cells []. This suggests that the cells have originated from a common origin, disseminating through the

41 Page 0 of vascular and lymphatic systems []. This also may explain the recurrence of LAM cells in the healthy lungs received by LAM patients post lung transplant [-]. In vitro studies by Goncharova et al. have also provided supporting evidence that a functional TSC/TSC complex acts to regulate normal cell migration through the mtorc pathway, which controls RhoA-GTPase activity []. The mtorc inhibitor rapamycin, a bacterial macrolide with immunosuppressive and antitumour properties, has been a major focus of LAM research. In vitro studies have shown rapamycin to effectively inhibit LAM cell proliferation [,,]. In addition, in the Multicenter International LAM Efficacy of Sirolimus (MILES) trial, McCormack et al. reported that patients with LAM treated with sirolimus (rapamycin) experienced stabilised lung function with a reduction in symptoms and improvement in quality of life compared to patients who were taking a placebo []. However limitations were present, most notably the continued decline in lung function following the discontinuation of sirolimus []. Similarly, in a murine model of TSC-null tumours, rapamycin inhibited tumour growth but its withdrawal resulted in TSC-null tumour regrowth together with a decreased survival []. Although the long term effects of rapamycin are not known in LAM, chronic sirolimus use has previously been associated with altered lipid and glucose metabolism resulting in hyperlipidemia, glucose intolerance, diabetes like syndromes and cancer [-]. Together, these in vitro and in vivo studies highlight that although rapamycin shows beneficial effects in the treatment of LAM, its requirement for long term use and its potential adverse effects highlight the need for alternative treatments. Since enhanced migration is a prominent feature of TSC-null cells, the lack of effect of rapamycin on migration [] suggests that mtorc inhibitors alone may not be the optimal

42 Page of Journal of Cellular and Molecular Medicine treatment for LAM. Alternative treatments, in particular, drugs that tackle the increased migratory capacity of LAM cells may provide added benefit. Since LAM is a disease characterised by enhanced cell proliferation and migration, where both processes occur, the examination of the effects of drugs on both processes is necessary. Moses et al. demonstrated that doxycycline, a second-generation tetracycline antibiotic, improved lung function and quality of life in a single LAM patient, with minimal side-effects []. In addition, we have previously reported that doxycycline can reduce the mitochondrial activity and extracellular levels of active MMP- in human LAM cells and TSC-null mouse embryonic fibroblasts (MEF) []. Furthermore, doxycycline has been shown to inhibit migration of cells in the development of arterial intimal lesions [], breast carcinoma [] and human melanoma [0]. Since the loss of TSC plays a prominent role in the phenotypic characteristics in LAM, the study of cells that are well characterised for TSC dysfunction is highly relevant. Examples of these cells are the TSC knockout mouse embryonic fibroblasts (MEF) [], MEF in which TSC is stably reintroduced [] and the Eker rat uterine leiomyoma (ELT) cells in which TSC is naturally absent []. In this study, we investigated the effects of doxycycline on the migratory capacity of cells deficient for TSC, identified the signalling pathways through which doxycycline works and examined the potential of combining doxycycline with the mtorc inhibitor rapamycin, to inhibit the enhanced migration, proliferation and wound closure of TSC-deficient cells.

43 Page of Materials and Methods Cell Culture Littermate derived p knockout TSC-negative MEF, TSC-positive MEF and isogenic p positive -TSC-null MEF and -TSC-positive MEF (where TSC was stably reintroduced) (a gift from Dr. D. Kwiatkowski, Brigham and Women s Hospital Boston, MA, USA) and Eker rat uterine leiomyoma (ELT) cells that were spontaneously deficient in TSC (a gift from Dr. Cheryl Walker, MD Anderson Cancer Center, TX, USA) were cultured as previously described [,,,,]. MEF, MEF and ELT cell preparation For experiments with MEF and MEF, the cells were seeded at a density of x cells.cm - in high glucose DMEM (Life Technologies, Carlsbad, CA, USA) containing % foetal bovine serum (FBS - Glendarach Biological, Melbourne, VIC, Australia) and % penicillin-streptomycin (Life Technologies) for hours. The medium was then changed to DMEM containing 0. % FBS and % penicillin-streptomycin (serum-reduced DMEM) for hours. The cells were then used as indicated in the experiments. ELT cells were cultured in DF medium (supplemented with % FBS) as previously described [,,,]. ELT cells were seeded at a density of x cells.cm - in DF medium for hours, after which the medium was changed to serum free DF-basal medium for hours as previously described []. The cells were then used as indicated in the experiments.

44 Page of Journal of Cellular and Molecular Medicine Doxycycline, rapamycin and Y- preparation Doxycycline hyclate (Sigma-Aldrich, St. Louis, MO, USA) was freshly prepared in sterile distilled water at a stock concentration of 0 mm. Insolution rapamycin (Calbiochem, St. Louis, MO, USA) was diluted in sterile dimethyl sulfoxide (DMSO; Sigma-Aldrich) to a stock concentration of mm and stored at -0 C prior to use. Y- (Calbiochem) a ROCK inhibitor was diluted in sterile water to a stock concentration of mm and stored at - 0 C prior to use. For all experiments, doxycycline was used at a final concentration range of 00 fm - µm, rapamycin at nm (with respective DMSO vehicle controls) and Y- at 0. µm. For experiments in which the effects of combining doxycycline and rapamycin were assessed, various combinations of concentrations were used as indicated, with respective DMSO vehicle controls. Proliferation assay Cells that were seeded and placed under serum-reduced conditions in -well flat-bottom, tissue-culture treated polystyrene cell culture plates (BD Falcon, Becton Dickinson, Franklin Lakes, NJ, USA) as described above, were pretreated with doxycycline or rapamycin alone or in combination or with Y- in the presence of serum-reduced DMEM for minutes. The cells were subsequently stimulated with % FBS with or without drug for hours. Cells were then trypsinsed and proliferation was assessed by counting the number of viable cells (trypan blue exclusion) using a haemocytometer.

45 Page of Migration assay Cells that were seeded and placed in serum reduced conditions in cm tissue culture flasks (BD Falcon ) as described above were trypsinised, counted and resuspended in serumreduced DMEM. Cell migration was assessed using a transwell migration assay as previously described [,]. Briefly, cells in suspension were pre-treated with doxycycline or rapamycin alone or in combination, or with Y- (ROCK inhibitor) for minutes before seeding onto collagen type I ( µg/ml) coated cell culture inserts (.0 µm polyethylene terephthalate (PET) membrane (BD Falcon ) at a density of x cells per 0. cm. The cells were then allowed to migrate for hours towards a chemoattractant of % FBS at ⁰ C, % CO. Non-migrated cells were removed and migrated cells were fixed with % (v/v) paraformaldehyde (PFA), stained with 0. % (w/v) toludine blue (Sigma) containing 0. % (w/v) boric acid (Sigma) and imaged (Olympus BX0, Center Valley, PA, USA). Cell migration was measured by manually counting cells in five regions of 00 µm and expressed as an average. Wound Assay Cells that were seeded and placed under serum-reduced conditions in -well flat-bottom polystyrene cell culture plates precoated with collagen type I ( µg/ml) as described above, were pretreated with doxycycline or rapamycin alone, or in combination for minutes prior to wounding. A wound was created by scratching the cell layer with a sterile 00 µl pipette tip (Thermo Fisher Scientific, Waltham, MA, USA). The cells were rinsed with serumreduced DMEM to remove any cell debris and freshly prepared drug treatments were replaced into each well. The cells were then incubated at C, % CO in a humidified incubation chamber (Clear State Solutions, Victoria, Australia) mounted on a Nikon Eclipse Ti-microscope (Nikon Eclipse Ti, Tokyo, Japan). Time-lapse images were captured at

46 Page of Journal of Cellular and Molecular Medicine intervals of hour for up to 0 hours as indicated and the rate of wound closure was assessed using Nikon Imaging software (NIS-Elements Imaging Software Version..0, Melville, NY, USA). Immunoblotting Levels of phosphorylated (phospho)-p0s kinase (p0sk), phospho-akt, phospho p/ MAPK (ERK/) and phospho-focal adhesion kinase (FAK) were measured using immunoblotting as previously described []. Briefly, cells that were seeded and placed in serum-reduced conditions in -well flat-bottom polystyrene cell culture plates were treated with doxycycline or rapamycin alone or in combination or with Y-. The cells were then lysed in modified RIPA buffer and the samples were size fractionated on % polyacrylamide gels and transferred to polyvinvylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA). The membranes were then blocked with % bovine serum albumin (BSA) diluted in phosphate buffered saline with 0.0% Tween-0 (PBS-T) and subsequently incubated with respective primary antibodies against phosphorylated proteins (as indicated in Table ) overnight at C, before incubation with respective immunoglobulin G- horseradish peroxidase (HRP) conjugated secondary antibody (as indicated in Table ) for hour at room temperature. The membranes were then visualised using chemiluminescence (Millipore, Temecula, CA, USA), imaged and analysed using a Kodak Image Station 000MM (Carestream Molecular Imaging Software Version.0.., Rochester, NY, USA). To allow for comparison of phosphorylated proteins to total proteins, antibodies were stripped from the membranes, the membranes were re-blocked and re-probed as previously described with either primary antibodies against total proteins (as indicated in Table ) or

47 Page of mouse anti-α-tubulin (loading control; :000 dilution in % PBS-T) (Santa Cruz Biotechnology, Santa Cruz, CA, USA) []. Respective immunoglobulin G- horseradish peroxidase (HRP) conjugated secondary antibodies (as indicated in Table ) were used. RhoA Activation Assay RhoA activity was measured using the G-LISA RhoA Activation Assay Biochem Kit as per the manufacturer's instructions (Cytoskeleton, CO, USA). Briefly, cells that were seeded and placed in serum-reduced conditions in cm tissue cell culture flasks were treated with or without Y-, rapamycin or doxycycline alone or in combination as indicated for minute (as assessed by time-course experiments to exhibit maximum RhoA expression levels). The cells were then lysed and the activity of RhoA was measured using the G-LISA assay at an absorbance of 0 nm with the limits of detection at 0.0 ng. Following the manufacturer's instructions, levels of active RhoA were then normalised to total Rho (-A, -B, -C) levels as measured using immunoblotting (as detailed above). Mouse monoclonal anti- Rho (-A, -B, -C) primary antibody (diluted :000 - Millipore) and goat polyclonal antimouse immunoglobulin G- HRP conjugated secondary antibody (diluted :000 - Dako) were used. Statistical analysis Results from n experiments were analysed using Student's unpaired t-test, area under the curve, repeated measures one-way analysis of variance (ANOVA) or repeated measures twoway ANOVA with a Bonferroni post-test where appropriate (Graphpad Prism Version, Graphpad Software Inc, La Jolla, CA, USA). A probability (p) value of less than or equal to 0.0 was considered statistically significant.

48 Page of Journal of Cellular and Molecular Medicine Results Enhanced cell migration and increased rate of wound closure in TSC-deficient cells In this study, we confirmed the enhanced migration of p knockout TSC-negative MEF compared to TSC-positive MEF (TSC-negative and TSC-positive, n=; p 0.0, Figure A) [,] and extended our findings to show, using -TSC-positive MEF in which TSC has been stably reintroduced, that cell migration was significantly lower in cells where TSC is present (-TSC-null and -TSC-positive MEF, n=; p 0.0, Figure A). In addition, the rate of wound closure in TSC-negative MEF was also significantly greater compared to TSC-positive MEF (area under the curve, TSC-negative and TSC-positive, n=; p 0.0, Figure B). Again, using -TSC-positive MEF, the rate at which the cells closed a wound was significantly decreased by the presence of TSC (-TSC-positive and -TSC-null, n=; p 0.0, Figure C). Doxycycline reduced enhanced cell migration in TSC-deficient cells Doxycycline (0 pm - µm) decreased % FBS-induced migration of TSC-negative MEF by % (TSC-negative, n=; p 0.0, Figure A) and of -TSC-null MEF by.. % (-TSC-null MEF, n=; p 0.0, Figure B). Doxycycline also decreased the rate of wound closure in both TSC-negative MEF and -TSC-null MEF ( µm - µm area under the curve, TSC-negative and -TSC-null MEF, n=; p 0.0, Figure C, D). Similarly, the ROCK inhibitor, Y- ( - µm) decreased FBS-induced migration of TSC-negative MEF by. -.0 % (TSC-negative MEF, n=; p 0.0, Figure E). In contrast, rapamycin, had no effect on the migration of TSC-negative MEF and -TSC-null MEF at any of the concentrations tested ( - 00 nm) (data not shown) in accordance with previous studies [].

49 Page of Doxycycline and Y- had no effect on cell proliferation 0. % FBS and % FBS induced the proliferation of TSC-negative MEF to a greater extent than TSC-positive MEF (TSC-negative and TSC-positive, n=; p 0.0, Figure E) and this is in accordance with previous studies [,]. Cell proliferation of -TSC- positive MEF in the presence of % FBS was less than that in -TSC-null MEF (- TSC-positive and -TSC-null MEF, n=; p 0.0, Figure E). We have previously shown that doxycycline has no effect on the proliferation of TSC- positive or TSC-negative MEF [] and we were able to replicate these findings in - TSC-positive and -TSC-null MEF (-TSC-positive and -TSC-null MEF, n=; p>0.0, Figure E) and in ELT cells where doxycycline inhibited cell proliferation only at the highest concentration ( µm) (ELT cells, n=, p 0.0, Figure E). In addition, Y- had no effect on the proliferation of TSC-positive or TSC-negative MEF (TSC- positive and TSC-negative MEF, n=; p>0.0, Figure E). To confirm that proliferation in the above cell types can be inhibited, the mtorc inhibitor rapamycin was used as a positive control, showing a reduction in the proliferation of MEF, MEF and ELT cells (TSC-negative and TSC-positive, n=; p 0.0, Supplement Figure E, -TSC-positive and -TSC-null MEF, n=; p 0.0, Figure E and ELT, n=; p 0.0, Figure E). Furthermore, doxycycline and Y- had no effect on the phosphorylation of p0sk, (TSC-positive or TSC-negative, n=, p>0.0, doxycycline Figure A, B, Y- Figure C, D), whereas rapamycin inhibited the phosphorylation of p0sk (TSC-positive and TSC-negative, n=, p 0.0, Figure E, F).

50 Page of Journal of Cellular and Molecular Medicine Doxycycline inhibits RhoA activity and reduces phosphorylation of FAK in TSC- negative MEF To investigate the mechanism through which doxycycline inhibits the migration of TSC- deficient cells, we examined the activity of RhoA-GTPase and the phosphorylation of FAK, AKT and ERK/. Under basal conditions and in the presence of % FBS, TSC-negative MEF exhibited increased RhoA-GTPase activity compared to TSC-positive MEF in accordance with published studies [,]. In addition, in TSC-positive MEF, RhoA- GTPase activity was induced in the presence of % FBS, whereas this was constitutively active in TSC-negative MEF (TSC-positive and TSC-negative MEF, 0. % FBS and % FBS, p 0.0, Figure A). Doxycycline ( µm & µm) and Y- ( µm) reduced elevated RhoA-GTPase activity in TSC-negative MEF by. -. % and. % at minute (TSC-negative, doxycycline and Y-, n=, p 0.0, Figure B), while rapamycin (00 nm) had no effect (TSC-negative, rapamycin 00 nm, n=, p 0.0, Figure B). In addition, doxycycline had no effect on RhoA-GTPase activity in TSC-positive MEF (TSC-positive, n=, p>0.0, Figure C). The amount of phospho-fak was constitutively higher in TSC-negative MEF compared to TSC-positive MEF (n=, p 0.0 Figure ). Doxycycline reduced elevated levels of phospho-fak in TSC-negative MEF while it had no effect in TSC-positive MEF (TSC- negative MEF, n=, p 0.0 Figure ). Doxycycline also had no effect on the levels of phospho-akt (n=, p>0.0, Figure A, B) or phospho-erk/ (p/) (n=, p>0.0, Figure C, D) in TSC-positive or TSC-negative MEF.

51 Page 0 of Combined treatment of doxycycline and rapamycin inhibits the rate of wound closure in TSC-negative MEFs We next assessed the effectiveness of combining rapamycin with doxycycline in inhibiting the proliferation, migration and the ability to close a wound of TSC-null MEF, as well as its effect on RhoA-GTPase activity. Doxycycline or rapamycin alone or in combination had no effect on the proliferation of TSC-positive MEF (TSC-positive, n=; p>0.0, Figure A). Combining doxycycline and rapamycin significantly inhibited TSC-negative MEF proliferation, migration and RhoA-GTPase activity, however the degree of inhibition was no greater compared to the individual drugs alone (Cell proliferation, TSC-negative, n=; p>0.0, Figure B, cell migration, TSC-negative, n=; p>0.0, Figure A and RhoA activity, n=, p>0.0, Figure B). The combination of doxycycline and rapamycin at sub-maximal concentrations decreased the rate of wound closure in TSC-negative MEF when compared to the individual effects of doxycycline or rapamycin alone (TSC-negative, n=; p<0.0, Figures C).

52 Page of Journal of Cellular and Molecular Medicine Discussion A lack of treatment for LAM has prompted studies to further understand the pathogenic characteristics of the disease, in an attempt to pinpoint possible future therapeutic targets. In this study, we demonstrated the ability of doxycycline to reduce the enhanced migratory capacity of cells deficient for TSC through the inhibition of RhoA-GTPase activity and reduced levels of phospho-fak. In addition, we extended our research to examine the combination of doxycycline and rapamycin and showed this reduced the rate of wound closure and maintained the reduction in migration, proliferation and RhoA-GTPase activity in TSC-null cells. Since LAM is associated with TSC dysfunction, cells deficient for TSC such as MEF, - MEF and ELT cells, have been widely used to enhance our understanding of this disease []. The use of TSC-negative MEF and -TSC-null MEF in this study is of particular relevance to LAM as some LAM cells have been reported to be TSC negative [,,]. In addition, TSC-negative and -TSC-null MEF have also been shown to exhibit enhanced proliferation and migration, characteristics which are also shared by LAM cells [,,,0]. The dysfunction of TSC has been widely demonstrated to be associated with enhanced proliferation of LAM cells [,,,,,]. Many studies have focused on controlling this enhanced proliferation through the anti-proliferative properties of rapamycin (an mtorc inhibitor) [,,,]. However, it is as important to recognise that the loss of TSC function in LAM not only results in the hallmark manifestations of enhanced cell proliferation, but also enhanced cell migration. For this reason, we believe that targeting the increased migratory capacity of cells deficient for TSC may be beneficial.

53 Page of It is well established that TSC plays a prominent role in the regulation of cell migration [,,,,]. Previous studies that examined cell migration using a transwell migration assay have demonstrated enhanced migratory capacity in cells deficient for TSC. In this study, we were able to confirm these findings and extended the study to assess the rate at which TSC-null cells can close a wound. Here, we demonstrated that the rate of wound closure in cells deficient for TSC was significantly increased compared to TSC-positive cells. In this study, we have demonstrated for the first time that doxycycline can reduce the migratory capabilities and reduce the rate of wound closure of TSC-negative MEF and - TSC-null MEF. It was of great interest that doxycycline inhibited cell migration at concentrations as low as 0 pm whereas it had no effect on the proliferation of -TSC- positive and -TSC-null MEF. This is in accordance with our previous study that showed doxycycline had no effect on the proliferation of TSC-positive, TSC-null MEF or human LAM cells []. Also in the present study, we showed that proliferation of ELT cells was inhibited by doxycycline only at high concentrations and these findings are similar to those of Chang et al. who reported that doxycycline at concentrations of - 0 µm [- µg.ml - ] had no effect on the proliferation of ELT cells but concentrations > µm [> µg.ml - ] decreased proliferation, increased apoptosis and altered cell morphology - effects which were due to doxycycline-induced toxicity []. Furthermore, we demonstrated rapamycin to be ineffective in the inhibition of migration and confirmed the findings of Goncharova et al. who showed rapamycin, at concentrations that significantly abrogated LAM cell proliferation, had no effect on human LAM cell migration []. Wound closure assays have previously been reported to reflect two crucial processes, cell proliferation and migration (Liang et al., 00). However, we have demonstrated in this study that, the ability of TSC-negative and -

54 Page of Journal of Cellular and Molecular Medicine TSC-null MEFs to close a wound relies predominantly on migration and not proliferation, as rapamycin had little effect in these assays. Matrix metalloproteinase (MMP) - and - have often been associated with cell migration and invasion; however the concentrations of doxycycline that inhibited cell migration were significantly lower than those we have previously reported for effective MMP- inhibition []. This suggests that, although doxycycline is capable of MMP- inhibition, the reduction in migratory capacity demonstrated in this study is not likely to be due to the inhibition of MMPs. It was also interesting to observe that doxycycline was not as effective in the wound closure assay as in the transwell migration assay, where concentrations of only 0 µm [µg.ml - ] significantly inhibited wound closure. This may be in part due to other processes involved in the wound closure assay such as cell-cell interactions. The rapamycin-insensitive mtorc pathway regulates cell migration through RhoA- GTPase, and, in this study, we confirmed previous findings that RhoA-GTPase activity in TSC-negative MEF was higher than in TSC-positive MEF under basal conditions and in the presence of % FBS []. The mechanism of action through which doxycycline inhibits TSC-null cell migration was not known, however this study shows doxycycline inhibits the migration of TSC-null cells through the activity of RhoA-GTPase, whereas levels of phospho-p0sk, phospho-akt and phospho-erk/ were unaltered. However, the mechanism by which doxycycline inhibits cell migration at the lower concentrations at which RhoA-GTPase activity was not affected (0 pm - 00 nm), is likely to be due to targeting other signalling pathways.

55 Page of Importantly, doxycycline inhibited the activity of RhoA-GTPase in TSC-negative MEF, while it was unaltered in TSC-positive MEF. RhoA-GTPase is known to play a prominent role in the regulation and organisation of the cytoskeleton by promoting the assembly of focal adhesions and by activating focal adhesion kinase (FAK) [,], processes which are important for cell motility. We have shown, in this study, that the protein tyrosine kinase FAK is constitutively active in TSC-negative MEF compared to TSC-positive MEF, thus supporting the finding of enhanced cell migration in TSC-negative MEF. Furthermore, doxycycline significantly inhibited levels of phospho-fak in TSC-negative MEF while they were unaltered in TSC-positive MEF. These data show that in TSC-negative MEF, doxycycline inhibited the activity of RhoA-GTPase at minute. This inhibition is then transduced downstream where FAK signalling was inhibited at hours and subsequently reduced functional migration at hours. The finding that doxycycline can regulate cell migration and FAK expression supports the findings by Sun et al. who showed doxycycline inhibited the adhesion and migration of melanoma cells through the inhibition of FAK expression []. Although no studies have demonstrated the sustained effects of doxycycline in the reduction of RhoA-GTPase activity, Sun et al. reported the effects of doxycycline in wound healing assays and FAK expression in the continued presence of doxycycline up to hours []. This suggests that the abrupt changes in RhoA-GTPase activity by doxycycline can influence outcomes of FAK expression and functional migration over a sustained period of time. It is now accepted that rapamycin alone may not be the most effective way to treat LAM, and investigations into combination therapies are underway [,]. Here, we explored the effects of combining doxycycline and rapamycin on the migration, proliferation, wound closure and RhoA activity of cells deficient for TSC. Although combining doxycycline and rapamycin

56 Page of Journal of Cellular and Molecular Medicine in this study inhibited those cellular parameters, the degree of inhibition was no greater than that of the individual drugs alone. The absence of a greater effect from the combination treatment was not surprising, as each drug works to inhibit independent functional characteristics and signalling pathways. However, it is important to note that the combination of doxycycline and rapamycin was not detrimental to the individual outcomes, i.e. it did not reduce the degree of inhibition caused by the individual drug treatment. Furthermore, as shown in the wound closure assay, combination treatment with submaximal doxycycline and rapamycin concentrations significantly reduced the rate of wound closure, whereas no effect was observed with individual doxycycline and rapamycin treatments at those concentrations. The mechanisms through which combination therapy reduced wound closure in TSC- negative MEF were not investigated in this study. The results from this study suggest that doxycycline may be of potential therapeutic benefit as a treatment for diseases in which TSC dysfunction results in enhanced cellular migration. In addition, this study provides preliminary evidence that the combination of doxycycline and rapamycin may lower the dosing requirement for rapamycin, and in turn potentially reduce the side effects associated with chronic rapamycin use. 0

57 Page of Acknowledgements The authors would like to acknowledge the financial contributions from LAM Australasia Research Alliance (LARA), Cooperative Research Centre (CRC) for Asthma and Airways, The New Zealand LAM Charitable Trust, The LAM Foundation and NIH/NHLBIROHL00 (to VPK) B.G.G. Oliver was supported by a NHMRC Career Development Fellowship #0. J,K. Burgess was supported by a NHMRC Career Development Fellowship #. J.L. Black was supported by a NHMRC Senior Principal Fellowship #. We thank Dr. David Kwiatkowski for providing the MEFs and Dr. Cheryl Walker for providing the ELT cells. Conflicts of interest The authors have no conflict of interest to declare

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59 Page of Goncharova EA, Goncharov DA, Spaits M, et al. Abnormal growth of smooth muscle-like cells in lymphangioleiomyomatosis: Role for tumor suppressor TSC. Am J Respir Cell Mol Biol. 00; : -.. Jacinto E, Loewith R, Schmidt A, et al. Mammalian TOR complex controls the actin cytoskeleton and is rapamycin insensitive. Nat Cell Biol. 00; : Sarbassov DD, Ali SM, Kim DH, et al. Rictor, a novel binding partner of mtor, defines a rapamycin-insensitive and raptor-independent pathway that regulates the cytoskeleton. Curr Biol. 00; : -.. Goncharova EA, Goncharov DA, Li H, et al. mtorc is required for proliferation and survival of TSC-null cells. Mol Cell Biol. 0; : -.. Yu J, Astrinidis A, Henske EP. Chromosome loss of heterozygosity in tuberous sclerosis and sporadic lymphangiomyomatosis. Am J Respir Crit Care Med. 00; : -0.. Crooks DM, Pacheco-Rodriguez G, DeCastro RM, et al. Molecular and genetic analysis of disseminated neoplastic cells in lymphangioleiomyomatosis. Proc Natl Acad Sci U S A. 00; : -.. Bittmann I, Dose TB, Muller C, et al. Lymphangioleiomyomatosis: recurrence after single lung transplantation. Hum Pathol. ; : 0-.. Bittmann I, Rolf B, Amann G, et al. Recurrence of lymphangioleiomyomatosis after single lung transplantation: new insights into pathogenesis. Hum Pathol. 00; : -.. Chen F, Bando T, Fukuse T, et al. Recurrent lymphangioleiomyomatosis after living-donor lobar lung transplantation. Transplant Proc. 00; : -.. Karbowniczek M, Astrinidis A, Balsara BR, et al. Recurrent lymphangiomyomatosis after transplantation: genetic analyses reveal a metastatic mechanism. Am J Respir Crit Care Med. 00; : -.. Nine JS, Yousem SA, Paradis IL, et al. Lymphangioleiomyomatosis: recurrence after lung transplantation. J Heart Lung Transplant. ; : -.. Barbet NC, Schneider U, Helliwell SB, et al. TOR controls translation initiation and early G progression in yeast. Mol Biol Cell. ; : -.. Black JL, Ge Q, Boustany S, et al. In vitro studies of lymphangioleiomyomatosis. Eur Respir J. 00; : -.. McCormack FX, Inoue Y, Moss J, et al. Efficacy and safety of sirolimus in lymphangioleiomyomatosis. N Engl J Med. 0; : -0.. Cruzado JM. Nonimmunosuppressive effects of mammalian target of rapamycin inhibitors. Transplant Rev (Orlando). 00; : -.. Morrisett JD, Abdel-Fattah G, Hoogeveen R, et al. Effects of sirolimus on plasma lipids, lipoprotein levels, and fatty acid metabolism in renal transplant patients. J Lipid Res. 00; : -0.. Stallone G, Infante B, Grandaliano G, et al. Management of side effects of sirolimus therapy. Transplantation. 00; : S-.. Teutonico A, Schena PF, Di Paolo S. Glucose metabolism in renal transplant recipients: effect of calcineurin inhibitor withdrawal and conversion to sirolimus. J Am Soc Nephrol. 00; : -.. Moses MA, Harper J, Folkman J. Doxycycline treatment for lymphangioleiomyomatosis with urinary monitoring for MMPs. N Engl J Med. 00; : -.

60 Page of Journal of Cellular and Molecular Medicine Moir LM, Ng HY, Poniris MH, et al. Doxycycline inhibits matrix metalloproteinase- secretion from TSC-null mouse embryonic fibroblasts and lymphangioleiomyomatosis cells. Br J Pharmacol. 0; : -.. Bendeck MP, Conte M, Zhang M, et al. Doxycycline modulates smooth muscle cell growth, migration, and matrix remodeling after arterial injury. Am J Pathol. 00; 0: -.. Fife RS, Sledge GW, Jr. Effects of doxycycline on in vitro growth, migration, and gelatinase activity of breast carcinoma cells. J Lab Clin Med. ; : Seftor RE, Seftor EA, De Larco JE, et al. Chemically modified tetracyclines inhibit human melanoma cell invasion and metastasis. Clin Exp Metastasis. ; : -.. Zhang H, Bajraszewski N, Wu E, et al. PDGFRs are critical for PIK/Akt activation and negatively regulated by mtor. J Clin Invest. 00; : -.. Howe SR, Gottardis MM, Everitt JI, et al. Rodent model of reproductive tract leiomyomata. Establishment and characterization of tumor-derived cell lines. Am J Pathol. ; : -.. Goncharova E, Goncharov D, Noonan D, et al. TSC modulates actin cytoskeleton and focal adhesion through TSC-binding domain and the Rac GTPase. J Cell Biol. 00; : -.. Moir LM, Trian T, Ge Q, et al. Phosphatidylinositol -kinase isoform-specific effects in airway mesenchymal cell function. J Pharmacol Exp Ther. 0; : -.. Zhang H, Cicchetti G, Onda H, et al. Loss of Tsc/Tsc activates mtor and disrupts PIK-Akt signaling through downregulation of PDGFR. J Clin Invest. 00; : -.. Finlay GA, Malhowski AJ, Liu Y, et al. Selective inhibition of growth of tuberous sclerosis complex null cells by atorvastatin is associated with impaired Rheb and Rho GTPase function and reduced mtor/s kinase activity. Cancer Res. 00; : -.. Kwiatkowski DJ. Animal models of lymphangioleiomyomatosis (LAM) and tuberous sclerosis complex (TSC). Lymphat Res Biol. 0; : -.. Hirama M, Atsuta R, Mitani K, et al. Lymphangioleiomyomatosis diagnosed by immunocytochemical and genetic analysis of lymphangioleiomyomatosis cell clusters found in chylous pleural effusion. Intern Med. 00; : -.. Darling TN, Pacheco-Rodriguez G, Gorio A, et al. Lymphangioleiomyomatosis and TSC-/- cells. Lymphat Res Biol. 0; : Goncharova EA, Ammit AJ, Irani C, et al. PIK is required for proliferation and migration of human pulmonary vascular smooth muscle cells. Am J Physiol Lung Cell Mol Physiol. 00; : L-.. Yu JJ, Robb VA, Morrison TA, et al. Estrogen promotes the survival and pulmonary metastasis of tuberin-null cells. Proc Natl Acad Sci U S A. 00; : -0.. Yu J, Parkhitko AA, Henske EP. Mammalian target of rapamycin signaling and autophagy: roles in lymphangioleiomyomatosis therapy. Proc Am Thorac Soc. 0; : -.. Kwiatkowski DJ, Manning BD. Tuberous sclerosis: a GAP at the crossroads of multiple signaling pathways. Hum Mol Genet. 00; Spec No. : R-.

61 Page 0 of Lesma E, Eloisa C, Isaia E, et al. Development of a lymphangioleiomyomatosis model by endonasal administration of human TSC-/- smooth muscle cells in mice. Am J Pathol. 0; : -0.. Chang WY, Clements D, Johnson SR. Effect of doxycycline on proliferation, MMP production, and adhesion in LAM-related cells. Am J Physiol Lung Cell Mol Physiol. 0; : L-00.. Chen CS, Alonso JL, Ostuni E, et al. Cell shape provides global control of focal adhesion assembly. Biochem Biophys Res Commun. 00; : -.. Lamb RF, Roy C, Diefenbach TJ, et al. The TSC tumour suppressor hamartin regulates cell adhesion through ERM proteins and the GTPase Rho. Nat Cell Biol. 000; : -.. Sun T, Zhao N, Ni CS, et al. Doxycycline inhibits the adhesion and migration of melanoma cells by inhibiting the expression and phosphorylation of focal adhesion kinase (FAK). Cancer Lett. 00; : -0.

62 Page of Journal of Cellular and Molecular Medicine Figure Legends Figure : % FBS-induced migration of A. TSC-positive (n=) and TSC-negative (n=) MEF and -TSC-positive MEF (n=) and -TSC-null MEF (n=). Data represent the average number of cells migrated in five regions of 00 µm ± SEM *p 0.0 unpaired t-test. Wound closure assay of B. TSC-positive (black line, n=) and TSC- negative MEF (red line, n=) over 0 hours and C. -TSC-positive (black line, n=) and -TSC-null MEF (red line, n=) over 0 hours. Data expressed as a percentage closure ± SEM in response to0. % FBS stimulation *p 0.0 repeated measures one-way ANOVA with a Bonferroni post test. Figure : Migration of A. TSC-negative MEF (n=) and B. -TSC-null MEF following minutes pretreatment with doxycycline. Data expressed as the average number of cells migrated in five regions of 00 µm ± SEM. Wound closure assay of C. TSC-negative MEF (n=) and D. -TSC-null MEF (n=) following minutes pretreatment with doxycycline. Data expressed as area under the curve (AUC) over 0 hours ± SEM.. Migration of E. TSC- negative MEF (n=) following minutes pretreatment with Y-. Data expressed as the average number of cells migrated per 00 µm *p 0.0 repeated measures one-way ANOVA compared with FBS with a Bonferroni post-test. Figure : Phospho-p0SK levels in A. TSC-positive MEF (n=) and B. TSC-negative MEF (n=) in the presence (+) or absence (-) of doxycycline as indicated, C. TSC-positive MEF (n=) and D. TSC-negative MEF (n=) in the presence (+) or absence (-) of Y- as indicated and E. TSC-positive MEF (n=) and F. TSC-negative MEF (n=) in the presence (+) or absence (-) of vehicle control (V) or rapamycin as indicated. Representative

63 Page of western blots of phospho-p0sk, p0sk and α-tubulin are shown (A-F) above mean data. Data expressed as mean ± SEM *p 0.0 repeated measures one-way ANOVA with a Bonferroni post-test (compared with % FBS). Figure : RhoA-GTPase activity in A. TSC-positive (n=) and TSC-negative MEF (n=) under basal conditions (0. % FBS) and in the presence of the stimulus % FBS # p 0.0 TSC-positive vs TSC-negative cells, B. TSC-negative MEF (n=) following minute of treatment with doxycycline, vehicle control (DMSO), rapamycin or Y- as indicated and C. TSC-positive MEF (n=) following minute of treatment with doxycycline, or Y-. RhoA activity corrected for total Rho (-A, -B, -C) and expressed as a percentage of % FBS ± SEM *p 0.0 repeated measures one-way ANOVA with a Bonferroni post-test (compared with % FBS). Figure : Phospho-FAK levels in TSC-positive and TSC-negative MEF (n=) in the presence (+) or absence (-) of doxycycline treatment for hours. Representative western blots of phospho-fak, FAK and α-tubulin are shown above mean data. Data expressed as mean ± SEM #p 0.0 compared with TSC-negative cells; *p 0.0 repeated measures oneway ANOVA with a Bonferroni post-test (compared with % FBS). Figure : Phospho-AKT levels in A. TSC-positive MEF (n=) and B. TSC-negative MEF (n=) and phospho-erk/ (p/) levels in C. TSC-positive MEF (n=) and D. TSC- negative MEF (n=) in the presence (+) or absence (-) of doxycycline as indicated. Representative western blots of phospho-akt, AKT, phospho-erk/ (p/), ERK/ (p/) and α-tubulin are shown above mean data. Data expressed as mean ± SEM *p 0.0

64 Page of Journal of Cellular and Molecular Medicine repeated measures one-way ANOVA with a Bonferroni post-test (comparison with % FBS). Figure : Proliferation of A. TSC-positive MEF (n=) and B. TSC-negative MEF (n=) treated with doxycycline, vehicle or rapamycin alone, or in combination as indicated. Data expressed as mean ± SEM *p 0.0 repeated measures one-way ANOVA compared to FBS #p 0.0 repeated measures one-way ANOVA compared to Vehicle (DMSO) control where appropriate. Bonferroni post-test was used. Figure : Cell migration of A. TSC-negative MEF (n=), treated with doxycycline (D) vehicle or rapamycin (R) alone, or in combination (R+D). Data expressed as mean ± SEM *p 0.0 comparison with % FBS, $ p<0.0 comparison with vehicle control, using repeated measures one-way ANOVA. B. RhoA activity of TSC-negative MEF (n=) treated with doxycycline(d), vehicle or rapamycin (R) alone or in combination. RhoA activity corrected for total Rho (-A, -B, -C) and expressed as a percentage of % FBS ± SEM *p 0.0 comparison with % FBS, $ p<0.0 comparison with vehicle control using repeated measures one-way ANOVA. C. Wound closure of TSC-negative MEF (n=) treated with doxycycline, vehicle or rapamycin nm alone or in combination. Data expressed as area under the curve (AUC) over 0 hours ± SEM. *p 0.0 comparison with % FBS, $ p<0.0 comparison with vehicle control, analysed using repeated measures one-way ANOVA as appropriate. Supplement Figure E. Basal level (0. % FBS) and % FBS-induced proliferation of TSC-positive (n=) and TSC-negative (n=) MEF. Data expressed as mean ± SEM *p 0.0 repeated measures two-way ANOVA with a Bonferroni post test.

65 Page of Supplement Figure E. Basal level (0. % FBS) and % FBS-induced proliferation of -TSC-positive (n=) and -TSC-null MEF (n=). Data expressed as mean ± SEM *p 0.0 repeated measures two-way ANOVA with a Bonferroni post test. Supplement Figure E. Proliferation of -TSC-positive (n=) and -TSC-null MEF (n=), treated with doxycycline. Data expressed as mean ± SEM *p 0.0 repeated measures one-way ANOVA with a Bonferroni post test. Supplement Figure E. Proliferation of ELT cells treated with doxycycline. Data expressed as mean ± SEM *p 0.0 compared to % FBS using repeated measures one-way ANOVA with a Bonferroni post test. Supplement Figure E. Proliferation of TSC-positive (white bars, n=) and TSC-negative MEF (grey bars, n=), treated with Y-. Data expressed as mean ± SEM *p 0.0 repeated measures one-way ANOVA with a Bonferroni post test. Supplement Figure E: Proliferation of TSC-positive (n=) and TSC-negative MEF (n=) treated with vehicle (V) or rapamycin. Data expressed as mean ± SEM # p 0.0 TSC- positive MEF compared to % FBS and *p 0.0 TSC-negative MEF compared to % FBS, repeated measures two-way ANOVA compared to DMSO vehicle control (V) with a Bonferroni post test. Supplement Figure E. Proliferation of -TSC-positive MEF (n=) and -TSC-null MEF (n=) treated with vehicle (V) or rapamycin. Data expressed as mean ± SEM #p 0.0

66 Page of Journal of Cellular and Molecular Medicine TSC-positive MEF compared to % FBS and *p 0.0 -TSC-null MEF compared to % FBS, repeated measures two-way ANOVA compared to DMSO vehicle control (V) with a Bonferroni post test. Supplement Figure E. Proliferation of ELT cells treated with vehicle (V) or rapamycin. Data expressed as mean ± SEM # p 0.0 ELT cells compared to % FBS repeated measures one-way ANOVA compared to DMSO vehicle control (V) with a Bonferroni post test.

67 Page of Table. Antibodies for immunoblotting Target Protein p0sk AKT p/ ERK/ MAPK FAK α-tubulin Primary Antibody / Secondary Antibody (Supplier) Concentration (Supplier) Rabbit polyclonal anti-mouse Phospho-p0SK (Thr ) / :000 (Cell Signalling Technology, / Danvers, MA, USA) Rabbit polyclonal anti-mouse p0sk / :000 (Cell Signalling Technology) Rabbit monoclonal anti-mouse Phospho-AKT (Thr ) / :000 (Cell Signalling Technology,) / Rabbit monoclonal anti-mouse AKT / :000 (Cell Signalling Technology,) Rabbit monoclonal anti-mouse Phospho-p/ MAPK (ERK/) (Thr 0 /Thr 0 ) / :000 (Cell Signalling Technology,) Rabbit monoclonal anti-mouse p/ MAPK (ERK/) / :000 (Cell Signalling Technology,) Goat polyclonal anti-rabbit immunoglobulin G- HRP conjugated :000 (Dako Glosturp, Denmark) Incubation Time (hrs) Rabbit polyclonal anti-mouse Phospho-FAK (Tyr ) / :000 (Cell Signalling Technology,) Rabbit polyclonal anti-mouse FAK / :000 (Cell Signalling Technology,) Mouse polyclonal α-tubulin :000 (SantaCruz) Goat polyclonal anti-mouse immunoglobulin G- HRP conjugated :000 (Dako) *Primary and secondary antibodies were diluted in % BSA/PBS-T. / -

68 Page of Journal of Cellular and Molecular Medicine Figure : % FBS-induced migration of A. TSC-positive (n=) and TSC-negative (n=) MEF and - TSC-positive MEF (n=) and -TSC-null MEF (n=). Data represent the average number of cells migrated in five regions of 00 µm ± SEM *p 0.0 unpaired t-test. Wound closure assay of B. TSC- positive (black line, n=) and TSC-negative MEF (red line, n=) over 0 hours and C. -TSC-positive (black line, n=) and -TSC-null MEF (red line, n=) over 0 hours. Data expressed as a percentage closure ± SEM in response to 0. % FBS stimulation *p 0.0 repeated measures one-way ANOVA with a Bonferroni post test. x0mm (0 x 0 DPI)

69 Page of Figure : Migration of A. TSC-negative MEF (n=) and B. -TSC-null MEF following minutes pretreatment with doxycycline. Data expressed as the average number of cells migrated in five regions of 00 µm± SEM. Wound closure assay of C. TSC-negative MEF (n=) and D. -TSC-null MEF (n=) following minutes pretreatment with doxycycline. Data expressed as area under the curve (AUC) over 0 hours ± SEM.. Migration of E. TSC-negative MEF (n=) following minutes pretreatment with Y-. Data expressed as the average number of cells migrated per 00 µm *p 0.0 repeated measures one-way ANOVA compared with FBS with a Bonferroni post-test. xmm (0 x 0 DPI)

70 Page of Journal of Cellular and Molecular Medicine Figure : Phospho-p0SK levels in A. TSC-positive MEF (n=) and B. TSC-negative MEF (n=) in the presence (+) or absence (-) of doxycycline as indicated, C. TSC-positive MEF (n=) and D. TSC-negative MEF (n=) in the presence (+) or absence (-) of Y- as indicated and E. TSC-positive MEF (n=) and F. TSC-negative MEF (n=) in the presence (+) or absence (-) of vehicle control (V) or rapamycin as indicated. Representative western blots of phospho-p0sk, p0sk and α-tubulin are shown (A-F) above mean data. Data expressed as mean ± SEM *p 0.0 repeated measures one-way ANOVA with a Bonferroni post-test (compared with % FBS). xmm (0 x 0 DPI)

71 Page 0 of Figure : RhoA-GTPase activity in A. TSC-positive (n=) and TSC-negative MEF (n=) under basal conditions (0. % FBS) and in the presence of the stimulus % FBS #p 0.0 TSC-positive vs TSC- negative cells, B. TSC-negative MEF (n=) following minute of treatment with doxycycline, vehicle control (DMSO), rapamycin or Y- as indicated and C. TSC-positive MEF (n=) following minute of treatment with doxycycline, or Y-. RhoA activity corrected for total Rho (-A, -B, -C) and expressed as a percentage of % FBS ± SEM *p 0.0 repeated measures one-way ANOVA with a Bonferroni post-test (compared with % FBS). 0xmm (00 x 00 DPI)

72 Page of Journal of Cellular and Molecular Medicine Figure : Phospho-FAK levels in TSC-positive and TSC-negative MEF (n=) in the presence (+) or absence (-) of doxycycline treatment for hours. Representative western blots of phospho-fak, FAK and α-tubulin are shown above mean data. Data expressed as mean ± SEM #p 0.0 compared with TSC-negative cells; *p 0.0 repeated measures one-way ANOVA with a Bonferroni post-test (compared with % FBS). 0x0mm (0 x 0 DPI)

73 Page of Figure : Phospho-AKT levels in A. TSC-positive MEF (n=) and B. TSC-negative MEF (n=) and phospho-erk/ (p/) levels in C. TSC-positive MEF (n=) and D. TSC-negative MEF (n=) in the presence (+) or absence (-) of doxycycline as indicated. Representative western blots of phospho-akt, AKT, phospho-erk/ (p/), ERK/ (p/) and α-tubulin are shown above mean data. Data expressed as mean ± SEM *p 0.0 repeated measures one-way ANOVA with a Bonferroni post-test (comparison with % FBS). xmm (0 x 0 DPI)

74 Page of Journal of Cellular and Molecular Medicine Figure : Proliferation of A. TSC-positive MEF (n=) and B. TSC-negative MEF (n=) treated with doxycycline, vehicle or rapamycin alone, or in combination as indicated. Data expressed as mean ± SEM *p 0.0 repeated measures one-way ANOVA compared to FBS #p 0.0 repeated measures one-way ANOVA compared to Vehicle (DMSO) control where appropriate. Bonferroni post-test was used. xmm (0 x 0 DPI)

75 Page of Figure : Cell migration of A. TSC-negative MEF (n=), treated with doxycycline (D) vehicle or rapamycin (R) alone, or in combination (R+D). Data expressed as mean ± SEM *p 0.0 comparison with % FBS, $p 0.0 comparison with vehicle control, using repeated measures one-way ANOVA. B. RhoA activity of TSC-negative MEF (n=) treated with doxycycline(d), vehicle or rapamycin (R) alone or in combination. RhoA activity corrected for total Rho (-A, -B, -C) and expressed as a percentage of % FBS ± SEM *p 0.0 comparison with % FBS, $p 0.0 comparison with vehicle control using repeated measures oneway ANOVA. C. Wound closure of TSC-negative MEF (n=) treated with doxycycline, vehicle or rapamycin nm alone or in combination. Data expressed as area under the curve (AUC) over 0 hours ± SEM. *p 0.0 comparison with % FBS, $p 0.0 comparison with vehicle control, analysed using repeated measures one-way ANOVA as appropriate. xmm (0 x 0 DPI)

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